Antisense modulation of PPP2R1A expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of PPP2R1A. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding PPP2R1A. Methods of using these compounds for modulation of PPP2R1A expression and for treatment of diseases associated with expression of PPP2R1A are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods formodulating the expression of PPP2R1A. In particular, this inventionrelates to compounds, particularly oligonucleotides, specificallyhybridizable with nucleic acids encoding PPP2R1A. Such compounds havebeen shown to modulate the expression of PPP2R1A.

BACKGROUND OF THE INVENTION

[0002] The adequate control of cellular growth and differentiation is aprerequisite for the proper development and functioning of highereukaryotic organisms. Extracellular molecules, such as hormones andgrowth factors, are important agents in determining this control. Thegenetic response of cells to these molecules requires signal receivers(receptors), signal transducers (second and third messengers), andsignal converters (transcription factors) that subsequently stimulate orrepress the transcription of target genes. As a consequence, the alteredpattern of gene expression will generate the respective phenotypicresponse, such as cell proliferation, differentiation, or apoptosis. Theimportance of appropriate regulation of these signal transductionpathways has been emphasized by the finding that many components ofthese networks are products of proto-oncogenes. If mutated orinappropriately expressed, they become oncoproteins that are able toconstitutively activate these pathways in the absence of externalstimuli, and thus are able to promote unrestricted cellularproliferation, which eventually may lead to tumorigenesis and cancer(Sontag, Cell. Signal., 2001, 13, 7-16).

[0003] Protein phosphatase 2A (PP2A) comprises a family ofserine/threonine phosphatases, minimally containing a well-conservedcatalytic subunit, the activity of which is highly regulated. Regulationis accomplished mainly by members of a family of regulatory subunits,which determine the substrate specificity, (sub)cellular localizationand catalytic activity of the PP2A holoenzymes. PP2A plays a prominentrole in the regulation of specific signal transduction cascades, aswitnessed by its presence in a number of macromolecular signalingmodules, where it is often found in association with other phosphatasesand kinases. Additionally, PP2A interacts with a substantial number ofother cellular and viral proteins, which are PP2A substrates, targetPP2A to different subcellular compartments or affect enzyme activity.Deregulation of PP2A occurs in pathological conditions such as cancerand neurodegenerative diseases as well as viral and parasitic diseases(Janssens and Goris, Biochem. J., 2001, 353, 417-439; Sontag, Cell.Signal., 2001, 13, 7-16).

[0004] The core PP2A enzyme is a dimer (PP2AD), consisting of a 36-kDacatalytic subunit (PP2AC) and a regulatory subunit of molecular mass65-kDa, known as the A subunit. A third regulatory B subunit can beassociated with this core structure. At present, four different familiesof B subunits have been identified (Janssens and Goris, Biochem. J.,2001, 353, 417-439).

[0005] The A subunit of PP2A is a structural subunit that is tightlyassociated with PP2AC, forming a scaffold to which the appropriate Bsubunit can bind. Different B subunits interact via the same oroverlapping sites within the A subunit of the core dimer, which explainswhy binding of the B subunits is mutually exclusive. The two distinctisoforms of the A subunit of PP2A, alpha and beta, share 86% sequenceidentity and are ubiquitously expressed (Janssens and Goris, Biochem.J., 2001, 353, 417-439).

[0006] PPP2R1A is the designation of the alpha isoform of the PP2A Asubunit (it also known as PP2-A alpha, PR65-alpha, and proteinphosphatase 2 (formerly 2A) regulatory subunit A (PR 65) alpha isoform).PPP2R1A has been cloned and mapped to chromosome 19q13.4 (Hemmings etal., Biochemistry, 1990, 29, 3166-3173; Ruteshouser et al., Oncogene,2001, 20, 2050-2054).

[0007] The finding that rat fibroblasts overexpressing PPP2R1A becomemultinucleated indicates that aberrant levels of PPP2R1A may severelycompromise the functional activity of PP2A to regulate the cell cycle. Apossible mechanism through which this event could occur is viasequestration of the catalytic or variable subunits of a PP2A holoenzymeinvolved in cytokinesis (Wera et al., J. Biol. Chem., 1995, 270,21374-21381).

[0008] Calin et al. have reported mutations of PPP2R1A in breastcarcinoma, lung carcinoma and melanoma cell lines which could causealterations of the PP2A holoenzyme that initiate the tumorigenic process(Calin et al., Oncogene, 2000, 19, 1191-1195).

[0009] Thus, selective modulation of PPP2R1A expression and/or activitymay prove to be an appropriate point for therapeutic intervention inpathological conditions such as hyperproliferative and neurodegenerativedisorders as well as disorders arising from aberrant apoptosis.

[0010] Small molecule inhibitors of protein phosphatases such as PP2Aare well known in the art. Examples of such small molecule inhibitorsinclude okadaic acid, calyculin A, microcystin-LR, tautomycin, nodularinand cantharidin (Janssens and Goris, Biochem. J., 2001, 353, 417-439).

[0011] Antisense-PP2A transfectants have been employed to inhibit theexpression of PP2A in investigations of proliferation of human myelomacells, IL-6 signal transduction in Hep3B cells and hyphal growth inNeurospora crassa (Choi et al., Immunol. Lett., 1998, 61, 103-107; Kangand Choi, Cell. Immunol., 2001, 213, 34-44; Yatzkan et al., Mol. Gen.Genet., 1998, 259, 523-531).

[0012] Disclosed and claimed in PCT publication WO 99/55906 is a methodof inducing programmed cell death in a cell with an effective amount ofan antisense nucleic acid molecule complementary to an mRNA encodingPP2A or an effective amount of a phosphatase inhibitor (Woodgett et al.,1999).

[0013] To date, investigative strategies aimed at modulating PPP2R1Aactivity and/or expression have involved the use of small moleculeinhibitors, antisense RNA transfections and antisense nucleic acidmolecules. Consequently, there remains a long felt need for additionalagents capable of effectively inhibiting PPP2R1A function.

[0014] Antisense technology is emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of expression of PPP2R1A.

[0015] The present invention provides compositions and methods formodulating expression of PPP2R1A.

SUMMARY OF THE INVENTION

[0016] The present invention is directed to compounds, particularlyantisense oligonucleotides, which are targeted to a nucleic acidencoding PPP2R1A, and which modulate the expression of PPP2R1A.Pharmaceutical and other compositions comprising the compounds of theinvention are also provided. Further provided are methods of modulatingthe expression of PPP2R1A in cells or tissues comprising contacting saidcells or tissues with one or more of the antisense compounds orcompositions of the invention. Further provided are methods of treatingan animal, particularly a human, suspected of having or being prone to adisease or condition associated with expression of PPP2R1A byadministering a therapeutically or prophylactically effective amount ofone or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding PPP2R1A, ultimately modulating theamount of PPP2R1A produced. This is accomplished by providing antisensecompounds which specifically hybridize with one or more nucleic acidsencoding PPP2R1A. As used herein, the terms “target nucleic acid” and“nucleic acid encoding PPP2R1A” encompass DNA encoding PPP2R1A, RNA(including pre-mRNA and mRNA) transcribed from such DNA, and also cDNAderived from such RNA. The specific hybridization of an oligomericcompound with its target nucleic acid interferes with the normalfunction of the nucleic acid. This modulation of function of a targetnucleic acid by compounds which specifically hybridize to it isgenerally referred to as “antisense”. The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translocation of the RNA to sites within the cell which are distant fromthe site of RNA synthesis, translation of protein from the RNA, splicingof the RNA to yield one or more mRNA species, and catalytic activitywhich may be engaged in or facilitated by the RNA. The overall effect ofsuch interference with target nucleic acid function is modulation of theexpression of PPP2R1A. In the context of the present invention,“modulation” means either an increase (stimulation) or a decrease(inhibition) in the expression of a gene. In the context of the presentinvention, inhibition is the preferred form of modulation of geneexpression and mRNA is a preferred target.

[0018] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding PPP2R1A. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding PPP2R1A, regardless of the sequence(s) of such codons.

[0019] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0020] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0021] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. mRNA transcriptsproduced via the process of splicing of two (or more) mRNAs fromdifferent gene sources are known as “fusion transcripts”. It has alsobeen found that introns can be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0022] It is also known in the art that alternative RNA transcripts canbe produced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic andextronic regions.

[0023] Upon excision of one or more exon or intron regions or portionsthereof during splicing, pre-mRNA variants produce smaller “mRNAvariants”. Consequently, mRNA variants are processed pre-mRNA variantsand each unique pre-mRNA variant must always produce a unique mRNAvariant as a result of splicing. These mRNA variants are also known as“alternative splice variants”. If no splicing of the pre-mRNA variantoccurs then the pre-mRNA variant is identical to the mRNA variant.

[0024] It is also known in the art that variants can be produced throughthe use of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites.

[0025] Once one or more target sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0026] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable.

[0027] An antisense compound is specifically hybridizable when bindingof the compound to the target DNA or RNA molecule interferes with thenormal function of the target DNA or RNA to cause a loss of activity,and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed. It is preferred that the antisense compoundsof the present invention comprise at least 80% sequence complementarityto a target region within the target nucleic acid, moreover that theycomprise 90% sequence complementarity and even more comprise 95%sequence complementarity to the target region within the target nucleicacid sequence to which they are targeted. For example, an antisensecompound in which 18 of 20 nucleobases of the antisense compound arecomplementary, and would therefore specifically hybridize, to a targetregion would represent 90 percent complementarity. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using basic local alignmentsearch tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990,215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0028] Antisense and other compounds of the invention, which hybridizeto the target and inhibit expression of the target, are identifiedthrough experimentation, and representative sequences of these compoundsare hereinbelow identified as preferred embodiments of the invention.The sites to which these preferred antisense compounds are specificallyhybridizable are hereinbelow referred to as “preferred target regions”and are therefore preferred sites for targeting. As used herein the term“preferred target region” is defined as at least an 8-nucleobase portionof a target region to which an active antisense compound is targeted.While not wishing to be bound by theory, it is presently believed thatthese target regions represent regions of the target nucleic acid whichare accessible for hybridization.

[0029] While the specific sequences of particular preferred targetregions are set forth below, one of skill in the art will recognize thatthese serve to illustrate and describe particular embodiments within thescope of the present invention. Additional preferred target regions maybe identified by one having ordinary skill.

[0030] Target regions 8-80 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative preferred target regions are considered to be suitablepreferred target regions as well.

[0031] Exemplary good preferred target regions include DNA or RNAsequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred target regions (theremaining nucleobases being a consecutive stretch of the same DNA or RNAbeginning immediately upstream of the 5′-terminus of the target regionand continuing until the DNA or RNA contains about 8 to about 80nucleobases). Similarly good preferred target regions are represented byDNA or RNA sequences that comprise at least the 8 consecutivenucleobases from the 3′-terminus of one of the illustrative preferredtarget regions (the remaining nucleobases being a consecutive stretch ofthe same DNA or RNA beginning immediately downstream of the 3′-terminusof the target region and continuing until the DNA or RNA contains about8 to about 80 nucleobases). One having skill in the art, once armed withthe empirically-derived preferred target regions illustrated herein willbe able, without undue experimentation, to identify further preferredtarget regions. In addition, one having ordinary skill in the art willalso be able to identify additional compounds, including oligonucleotideprobes and primers, that specifically hybridize to these preferredtarget regions using techniques available to the ordinary practitionerin the art.

[0032] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

[0033] For use in kits and diagnostics, the antisense compounds of thepresent invention, either alone or in combination with other antisensecompounds or therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

[0034] Expression patterns within cells or tissues treated with one ormore antisense compounds are compared to control cells or tissues nottreated with antisense compounds and the patterns produced are analyzedfor differential levels of gene expression as they pertain, for example,to disease association, signaling pathway, cellular localization,expression level, size, structure or function of the genes examined.These analyses can be performed on stimulated or unstimulated cells andin the presence or absence of other compounds which affect expressionpatterns.

[0035] Examples of methods of gene expression analysis known in the artinclude DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000,480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999,20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80,143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (reviewed in To, Comb. Chem. High Throughput Screen, 2000, 3,235-41).

[0036] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisenseoligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. Antisenseoligonucleotide drugs, including ribozymes, have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

[0037] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0038] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about80 nucleobases (i.e. from about 8 to about 80 linked nucleosides).Particularly preferred antisense compounds are antisenseoligonucleotides from about 8 to about 50 nucleobases, even morepreferably those comprising from about 12 to about 30 nucleobases.Antisense compounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression.

[0039] Antisense compounds 8-80 nucleobases in length comprising astretch of at least eight (8) consecutive nucleobases selected fromwithin the illustrative antisense compounds are considered to besuitable antisense compounds as well.

[0040] Exemplary preferred antisense compounds include DNA or RNAsequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the same DNAor RNA beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the DNA or RNA contains about 8 toabout 80 nucleobases). Similarly preferred antisense compounds arerepresented by DNA or RNA sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the DNA or RNA contains about 8 to about 80 nucleobases). Onehaving skill in the art, once armed with the empirically-derivedpreferred antisense compounds illustrated herein will be able, withoutundue experimentation, to identify further preferred antisensecompounds.

[0041] Antisense and other compounds of the invention, which hybridizeto the target and inhibit expression of the target, are identifiedthrough experimentation, and representative sequences of these compoundsare herein identified as preferred embodiments of the invention. Whilespecific sequences of the antisense compounds are set forth herein, oneof skill in the art will recognize that these serve to illustrate anddescribe particular embodiments within the scope of the presentinvention. Additional preferred antisense compounds may be identified byone having ordinary skill.

[0042] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric structure can be further joined to form a circular structure,however, open linear structures are generally preferred. In addition,linear structures may also have internal nucleobase complementarity andmay therefore fold in a manner as to produce a double strandedstructure. Within the oligonucleotide structure, the phosphate groupsare commonly referred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

[0043] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0044] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be a basic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0045] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;5,672,697 and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

[0046] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0047] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

[0048] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0049] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0050] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′—O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0051] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos.: 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

[0052] A further preferred modification includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring thereby forming a bicyclic sugar moiety. Thelinkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

[0053] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

[0054] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205;5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588;6,005,096; and 5,681,941, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, which is commonly owned with theinstant application and also herein incorporated by reference.

[0055] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. The compounds of the inventioncan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugate groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluores-ceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992 theentire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937). Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

[0056] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0057] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,increased stability and/or increased binding affinity for the targetnucleic acid. An additional region of the oligonucleotide may serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNAse H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof oligonucleotide inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as interferon-induced RNAseL whichcleaves both cellular and viral RNA. Consequently, comparable resultscan often be obtained with shorter oligonucleotides when chimericoligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

[0058] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0059] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0060] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos.: 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0061] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal, including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0062] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0063] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0064] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

[0065] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0066] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of PPP2R1A is treated by administering antisense compounds inaccordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

[0067] The antisense compounds of the invention are useful for researchand diagnostics, because these compounds hybridize to nucleic acidsencoding PPP2R1A, enabling sandwich and other assays to easily beconstructed to exploit this fact. Hybridization of the antisenseoligonucleotides of the invention with a nucleic acid encoding PPP2R1Acan be detected by means known in the art. Such means may includeconjugation of an enzyme to the oligonucleotide, radiolabelling of theoligonucleotide or any other suitable detection means. Kits using suchdetection means for detecting the level of PPP2R1A in a sample may alsobe prepared.

[0068] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0069] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful. Preferred topical formulationsinclude those in which the oligonucleotides of the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Preferred lipids and liposomes include neutral (e.g.dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Oligonucleotides of the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters include but are not limited arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999 which is incorporated herein byreference in its entirety.

[0070] Compositions and formulations for oral administration includepowders or granules, microparticulates, nanoparticulates, suspensions orsolutions in water or non- aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Preferred oralformulations are those in which oligonucleotides of the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Preferred surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Preferred bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferredfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also preferred are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly preferred combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl etherOligonucleotides of the invention may be delivered orally, in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. application Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No.09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23,1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298(filed May 20, 1999), each of which is incorporated herein by referencein their entirety.

[0071] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0072] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0073] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0074] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0075] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

[0076] Emulsions

[0077] The compositions of the present invention may be prepared andformulated as emulsions. Emulsions are typically heterogenous systems ofone liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases, and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

[0078] Emulsions are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0079] Synthetic surfactants, also known as surface active agents, havefound wide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

[0080] Naturally occurring emulsifiers used in emulsion formulationsinclude lanolin, beeswax, phosphatides, lecithin and acacia. Absorptionbases possess hydrophilic properties such that they can soak up water toform w/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

[0081] A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0082] Hydrophilic colloids or hydrocolloids include naturally occurringgums and synthetic polymers such as polysaccharides (for example,acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, andtragacanth), cellulose derivatives (for example, carboxymethylcelluloseand carboxypropylcellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

[0083] Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

[0084] The application of emulsion formulations via dermatological, oraland parenteral routes and methods for their manufacture have beenreviewed in the literature (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199). Emulsion formulations for oral deliveryhave been very widely used because of ease of formulation, as well asefficacy from an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

[0085] In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0086] The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

[0087] Surfactants used in the preparation of microemulsions include,but are not limited to, ionic surfactants, non-ionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (S0750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

[0088] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

[0089] Microemulsions of the present invention may also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

[0090] Liposomes

[0091] There are many organized surfactant structures besidesmicroemulsions that have been studied and used for the formulation ofdrugs. These include monolayers, micelles, bilayers and vesicles.Vesicles, such as liposomes, have attracted great interest because oftheir specificity and the duration of action they offer from thestandpoint of drug delivery. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers.

[0092] Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

[0093] In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

[0094] Further advantages of liposomes include; liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

[0095] Liposomes are useful for the transfer and delivery of activeingredients to the site of action. Because the liposomal membrane isstructurally similar to biological membranes, when liposomes are appliedto a tissue, the liposomes start to merge with the cellular membranesand as the merging of the liposome and cell progresses, the liposomalcontents are emptied into the cell where the active agent may act.

[0096] Liposomal formulations have been the focus of extensiveinvestigation as the mode of delivery for many drugs. There is growingevidence that for topical administration, liposomes present severaladvantages over other formulations. Such advantages include reducedside-effects related to high systemic absorption of the administereddrug, increased accumulation of the administered drug at the desiredtarget, and the ability to administer a wide variety of drugs, bothhydrophilic and hydrophobic, into the skin.

[0097] Several reports have detailed the ability of liposomes to deliveragents including high-molecular weight DNA into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin. The majority of applicationsresulted in the targeting of the upper epidermis.

[0098] Liposomes fall into two broad classes. Cationic liposomes arepositively charged liposomes which interact with the negatively chargedDNA molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

[0099] Liposomes which are pH-sensitive or negatively-charged, entrapDNA rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

[0100] One major type of liposomal composition includes phospholipidsother than naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0101] Several studies have assessed the topical delivery of liposomaldrug formulations to the skin. Application of liposomes containinginterferon to guinea pig skin resulted in a reduction of skin herpessores while delivery of interferon via other means (e.g. as a solutionor as an emulsion) were ineffective (Weiner et al., Journal of DrugTargeting, 1992, 2, 405-410). Further, an additional study tested theefficacy of interferon administered as part of a liposomal formulationto the administration of interferon using an aqueous system, andconcluded that the liposomal formulation was superior to aqueousadministration (du Plessis et al., Antiviral Research, 1992, 18,259-265).

[0102] Non-ionic liposomal systems have also been examined to determinetheir utility in the delivery of drugs to the skin, in particularsystems comprising non-ionic surfactant and cholesterol. Non-ionicliposomal formulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

[0103] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,53, 3765).

[0104] Various liposomes comprising one or more glycolipids are known inthe art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0105] Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

[0106] A limited number of liposomes comprising nucleic acids are knownin the art. WO 96/40062 to Thierry et al. discloses methods forencapsulating high molecular weight nucleic acids in liposomes. U.S.Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomesand asserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

[0107] Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

[0108] Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes. The most common wayof classifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

[0109] If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

[0110] If the surfactant molecule carries,a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

[0111] If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

[0112] If the surfactant molecule has the ability to carry either apositive or negative charge, the surfactant is classified as amphoteric.Amphoteric surfactants include acrylic acid derivatives, substitutedalkylamides, N-alkylbetaines and phosphatides.

[0113] The use of surfactants in drug products, formulations and inemulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms,Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0114] Penetration Enhancers

[0115] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

[0116] Penetration enhancers may be classified as belonging to one offive broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Eachof the above mentioned classes of penetration enhancers are describedbelow in greater detail.

[0117] Surfactants: In connection with the present invention,surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of oligonucleotidesthrough the mucosa is enhanced. In addition to bile salts and fattyacids, these penetration enhancers include, for example, sodium laurylsulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetylether) (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p.92); and perfluorochemical emulsions, such as FC-43.Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0118] Fatty acids: Various fatty acids and their derivatives which actas penetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0119] Bile salts: The physiological role of bile includes thefacilitation of dispersion and absorption of lipids and fat-solublevitamins (Brunton, Chapter 38 in: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, NewYork, 1996, pp. 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus the term “bilesalts” includes any of the naturally occurring components of bile aswell as any of their synthetic derivatives. The bile salts of theinvention include, for example, cholic acid (or its pharmaceuticallyacceptable sodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0120] Chelating Agents: Chelating agents, as used in connection withthe present invention, can be defined as compounds that remove metallicions from solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0121] Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

[0122] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

[0123] Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

[0124] Carriers

[0125] Certain compositions of the present invention also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0126] Excipients

[0127] In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

[0128] Pharmaceutically acceptable organic or inorganic excipientsuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

[0129] Formulations for topical administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

[0130] Suitable pharmaceutically acceptable excipients include, but arenot limited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

[0131] Other Components

[0132] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

[0133] Aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

[0134] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

[0135] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

[0136] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0137] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1

[0138] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxyand 2′-alkoxy amidites

[0139] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylphosphoramidites were purchased from commercial sources (e.g. Chemgenes,Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxysubstituted nucleoside amidites are prepared as described in U.S. Pat.No. 5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, optimized synthesis cycles weredeveloped that incorporate multiple steps coupling longer wait timesrelative to standard synthesis cycles.

[0140] The following abbreviations are used in the text: thin layerchromatography (TLC), melting point (MP), high pressure liquidchromatography (HPLC), Nuclear Magnetic Resonance (NMR), argon (Ar),methanol (MeOH), dichloromethane (CH₂Cl₂), triethylamine (TEA), dimethylformamide (DMF), ethyl acetate (EtOAc), dimethyl sulfoxide (DMSO),tetrahydrofuran (THF).

[0141] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-dC)nucleotides were synthesized according to published methods (Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203) using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.) or prepared as follows:

[0142] Preparation of 5′-O-Dimethoxytrityl-thymidine intermediate for5-methyl dC amidite

[0143] To a 50 L glass reactor equipped with air stirrer and Ar gas linewas added thymidine (1.00 kg, 4.13 mol) in anhydrous pyridine (6 L) atambient temperature. Dimethoxytrityl (DMT) chloride (1.47 kg, 4.34 mol,1.05 eq) was added as a solid in four portions over 1 h. After 30 min,TLC indicated approx. 95% product, 2% thymidine, 5% DMT reagent andby-products and 2% 3′,5′-bis DMT product (R_(f) in EtOAc 0.45, 0.05,0.98, 0.95 respectively). Saturated sodium bicarbonate (4 L) and CH₂Cl₂were added with stirring (pH of the aqueous layer 7.5). An additional 18L of water was added, the mixture was stirred, the phases wereseparated, and the organic layer was transferred to a second 50 Lvessel. The aqueous layer was extracted with additional CH₂Cl₂ (2×2 L).The combined organic layer was washed with water (10 L) and thenconcentrated in a rotary evaporator to approx. 3.6 kg total weight. Thiswas redissolved in CH₂Cl₂ (3.5 L), added to the reactor followed bywater (6 L) and hexanes (13 L). The mixture was vigorously stirred andseeded to give a fine white suspended solid starting at the interface.After stirring for 1 h, the suspension was removed by suction through a½″ diameter teflon tube into a 20 L suction flask, poured onto a 25 cmCoors Buchner funnel, washed with water (2×3 L) and a mixture ofhexanes- CH₂Cl₂ (4:1, 2×3 L) and allowed to air dry overnight in pans(1″ deep). This was further dried in a vacuum oven (75° C., 0.1 mm Hg,48 h) to a constant weight of 2072 g (93%) of a white solid, (mp122-124° C.). TLC indicated a trace contamination of the bis DMTproduct. NMR spectroscopy also indicated that 1-2 mole percent pyridineand about 5 mole percent of hexanes was still present.

[0144] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidineIntermediate for 5-methyl-dC amidite

[0145] To a 50 L Schott glass-lined steel reactor equipped with anelectric stirrer, reagent addition pump (connected to an additionfunnel), heating/cooling system, internal thermometer and an Ar gas linewas added 5′-O-dimethoxytrityl-thymidine (3.00 kg, 5.51 mol), anhydrousacetonitrile (25 L) and TEA (12.3 L, 88.4 mol, 16 eq). The mixture waschilled with stirring to −10° C. internal temperature (external −20°C.). Trimethylsilylchloride (2.1 L, 16.5 mol, 3.0 eq) was added over 30minutes while maintaining the internal temperature below −5° C.,followed by a wash of anhydrous acetonitrile (1 L). Note: the reactionis mildly exothermic and copious hydrochloric acid fumes form over thecourse of the addition. The reaction was allowed to warm to 0° C. andthe reaction progress was confirmed by TLC (EtOAc-hexanes 4:1; R_(f)0.43 to 0.84 of starting material and silyl product, respectively). Uponcompletion, triazole (3.05 kg, 44 mol, 8.0 eq) was added the reactionwas cooled to −20° C. internal temperature (external −30° C.).Phosphorous oxychloride (1035 mL, 11.1 mol, 2.01 eq) was added over 60min so as to maintain the temperature between −20° C. and −10° C. duringthe strongly exothermic process, followed by a wash of anhydrousacetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1h. TLC indicated a complete conversion to the triazole product (R_(f)0.83 to 0.34 with the product spot glowing in long wavelength UV light).The reaction mixture was a peach-colored thick suspension, which turneddarker red upon warming without apparent decomposition. The reaction wascooled to −15° C. internal temperature and water (5 L) was slowly addedat a rate to maintain the temperature below +10° C. in order to quenchthe reaction and to form a homogenous solution. (Caution: this reactionis initially very strongly exothermic). Approximately one-half of thereaction volume (22 L) was transferred by air pump to another vessel,diluted with EtOAc (12 L) and extracted with water (2×8 L). The combinedwater layers were back-extracted with EtOAc (6 L). The water layer wasdiscarded and the organic layers were concentrated in a 20 L rotaryevaporator to an oily foam. The foam was coevaporated with anhydrousacetonitrile (4 L) to remove EtOAc. (note: dioxane may be used insteadof anhydrous acetonitrile if dried to a hard foam). The second half ofthe reaction was treated in the same way. Each residue was dissolved indioxane (3 L) and concentrated ammonium hydroxide (750 mL) was added. Ahomogenous solution formed in a few minutes and the reaction was allowedto stand overnight (although the reaction is complete within 1 h).

[0146] TLC indicated a complete reaction (product R_(f) 0.35 inEtOAc-MeOH 4:1). The reaction solution was concentrated on a rotaryevaporator to a dense foam. Each foam was slowly redissolved in warmEtOAc (4 L; 50° C.), combined in a 50 L glass reactor vessel, andextracted with water (2×4L) to remove the triazole by-product. The waterwas back-extracted with EtOAc (2 L). The organic layers were combinedand concentrated to about 8 kg total weight, cooled to 0° C. and seededwith crystalline product. After 24 hours, the first crop was collectedon a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc (3×3L)until a white powder was left and then washed with ethyl ether (2×3L).The solid was put in pans (1″ deep) and allowed to air dry overnight.The filtrate was concentrated to an oil, then redissolved in EtOAc (2L), cooled and seeded as before. The second crop was collected andwashed as before (with proportional solvents) and the filtrate was firstextracted with water (2×1L) and then concentrated to an oil. The residuewas dissolved in EtOAc (1 L) and yielded a third crop which was treatedas above except that more washing was required to remove a yellow oilylayer.

[0147] After air-drying, the three crops were dried in a vacuum oven(50° C., 0.1 mm Hg, 24 h) to a constant weight (1750, 600 and 200 g,respectively) and combined to afford 2550 g (85%) of a white crystallineproduct (MP 215-217° C.) when TLC and NMR spectroscopy indicated purity.The mother liquor still contained mostly product (as determined by TLC)and a small amount of triazole (as determined by NMR spectroscopy), bisDMT product and unidentified minor impurities. If desired, the motherliquor can be purified by silica gel chromatography using a gradient ofMeOH (0-25%) in EtOAc to further increase the yield.

[0148] Preparation of5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite

[0149] Crystalline 5′-O-dimethoxytrityl-5-methyl-2′-deoxycytidine (2000g, 3.68 mol) was dissolved in anhydrous DMF (6.0 kg) at ambienttemperature in a 50 L glass reactor vessel equipped with an air stirrerand argon line. Benzoic anhydride (Chem Impex not Aldrich, 874 g, 3.86mol, 1.05 eq) was added and the reaction was stirred at ambienttemperature for 8 h. TLC (CH₂Cl₂-EtOAc; CH₂Cl₂-EtOAc 4:1; R_(f) 0.25)indicated approx. 92% complete reaction. An additional amount of benzoicanhydride (44 g, 0.19 mol) was added. After a total of 18 h, TLCindicated approx. 96% reaction completion. The solution was diluted withEtOAc (20 L), TEA (1020 mL, 7.36 mol, ca 2.0 eq) was added withstirring, and the mixture was extracted with water (15 L, then 2×10 L).The aqueous layer was removed (no back-extraction was needed) and theorganic layer was concentrated in 2×20 L rotary evaporator flasks untila foam began to form. The residues were coevaporated with acetonitrile(1.5 L each) and dried (0.1 mm Hg, 25° C., 24 h) to 2520 g of a densefoam. High pressure liquid chromatography (HPLC) revealed acontamination of 6.3% of N4, 3′-O-dibenzoyl product, but very littleother impurities.

[0150] The product was purified by Biotage column chromatography (5 kgBiotage) prepared with 65:35:1 hexanes-EtOAc-TEA (4L). The crude product(800 g),dissolved in CH₂Cl₂ (2 L), was applied to the column. The columnwas washed with the 65:35:1 solvent mixture (20 kg), then 20:80:1solvent mixture (10 kg), then 99:1 EtOAc:TEA (17kg). The fractionscontaining the product were collected, and any fractions containing theproduct and impurities were retained to be resubjected to columnchromatography. The column was re-equilibrated with the original 65:35:1solvent mixture (17 kg). A second batch of crude product (840 g) wasapplied to the column as before. The column was washed with thefollowing solvent gradients: 65:35:1 (9 kg), 55:45:1 (20 kg), 20:80:1(10 kg), and 99:1 EtOAc:TEA(15 kg). The column was reequilibrated asabove, and a third batch of the crude product (850 g) plus impurefractions recycled from the two previous columns (28 g) was purifiedfollowing the procedure for the second batch. The fractions containingpure product combined and concentrated on a 20L rotary evaporator,co-evaporated with acetontirile (3 L) and dried (0.1 mm Hg, 48 h, 25°C.) to a constant weight of 2023 g (85%) of white foam and 20 g ofslightly contaminated product from the third run. HPLC indicated apurity of 99.8% with the balance as the diBenzoyl product.

[0151][5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite)

[0152]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidine(998 g, 1.5 mol) was dissolved in anhydrous DMF (2 L). The solution wasco-evaporated with toluene (300 ml) at 50° C. under reduced pressure,then cooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5g, 0.75 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (15 ml) was added and the mixture was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (2.5 L) and water (600 ml), and extracted with hexane(3×3 L). The mixture was diluted with water (1.2 L) and extracted with amixture of toluene (7.5 L) and hexane (6 L). The two layers wereseparated, the upper layer was washed with DMF-water (7:3 v/v, 3×2 L)and water (3×2 L), and the phases were separated. The organic layer wasdried (Na₂SO₄), filtered and rotary evaporated. The residue wasco-evaporated with acetonitrile (2×2 L) under reduced pressure and driedto a constant weight (25° C., 0.1 mm Hg, 40 h) to afford 1250 g anoff-white foam solid (96%).

[0153] 2′-Fluoro Amidites

[0154] 2′-Fluorodeoxyadenosine amidites

[0155] 2′-fluoro oligonucleotides were synthesized as describedpreviously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] andU.S. Pat. No. 5,670,633, herein incorporated by reference. Thepreparation of 2′-fluoropyrimidines containing a 5-methyl substitutionare described in U.S. Pat. No. 5,861,493. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and whereby the 2′-alpha-fluoro atom is introduced bya SN2-displacement of a 2′-beta-triflate group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyl groups was accomplished usingstandard methodologies to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

[0156] 2′-Fluorodeoxyguanosine

[0157] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate isobutyryl-arabinofuranosylguanosine. Alternatively,isobutyryl-arabinofuranosylguanosine was prepared as described by Rosset al., (Nucleosides & Nucleosides, 16, 1645, 1997). Deprotection of theTPDS group was followed by protection of the hydroxyl group with THP togive isobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0158] 2′-Fluorouridine

[0159] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

[0160] 2′-Fluorodeoxycytidine

[0161] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0162] 2′-O-(2-Methoxyethyl) modified amidites

[0163] 2′-O-Methoxyethyl-substituted nucleoside amidites (otherwiseknown as MOE amidites) are prepared as follows, or alternatively, as perthe methods of Martin, P., (Helvetica Chimica Acta, 1995, 78, 486-504).

[0164] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine intermediate

[0165] 2,2′-Anhydro-5-methyl-uridine (2000 g, 8.32 mol),tris(2-methoxyethyl)borate (2504 g, 10.60 mol), sodium bicarbonate (60g, 0.70 mol) and anhydrous 2-methoxyethanol (5 L) were combined in a 12L three necked flask and heated to 130° C. (internal temp) atatmospheric pressure, under an argon atmosphere with stirring for 21 h.TLC indicated a complete reaction. The solvent was removed under reducedpressure until a sticky gum formed (50-85° C. bath temp and 100-11 mmHg) and the residue was redissolved in water (3 L) and heated to boilingfor 30 min in order the hydrolyze the borate esters. The water wasremoved under reduced pressure until a foam began to form and then theprocess was repeated. HPLC indicated about 77% product, 15% dimer (5′ ofproduct attached to 2′ of starting material) and unknown derivatives,and the balance was a single unresolved early eluting peak.

[0166] The gum was redissolved in brine (3 L), and the flask was rinsedwith additional brine (3 L). The combined aqueous solutions wereextracted with chloroform (20 L) in a heavier-than continuous extractorfor 70 h. The chloroform layer was concentrated by rotary evaporation ina 20 L flask to a sticky foam (2400 g). This was coevaporated with MeOH(400 mL) and EtOAc (8 L) at 75° C. and 0.65 atm until the foam dissolvedat which point the vacuum was lowered to about 0.5 atm. After 2.5 L ofdistillate was collected a precipitate began to form and the flask wasremoved from the rotary evaporator and stirred until the suspensionreached ambient temperature. EtOAc (2 L) was added and the slurry wasfiltered on a 25 cm table top Buchner funnel and the product was washedwith EtOAc (3×2 L). The bright white solid was air dried in pans for 24h then further dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) toafford 1649 g of a white crystalline solid (mp 115.5-116.5° C.).

[0167] The brine layer in the 20 L continuous extractor was furtherextracted for 72 h with recycled chloroform. The chloroform wasconcentrated to 120 g of oil and this was combined with the motherliquor from the above filtration (225 g), dissolved in brine (250 mL)and extracted once with chloroform (250 mL). The brine solution wascontinuously extracted and the product was crystallized as describedabove to afford an additional 178 g of crystalline product containingabout 2% of thymine. The combined yield was 1827 g (69.4%). HPLCindicated about 99.5% purity with the balance being the dimer.

[0168] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridinepenultimate intermediate

[0169] In a 50 L glass-lined steel reactor,2′-O-(2-methoxyethyl)-5-methyl-uridine (MOE-T, 1500 g, 4.738 mol),lutidine (1015 g, 9.476 mol) were dissolved in anhydrous acetonitrile(15 L). The solution was stirred rapidly and chilled to −10° C.(internal temperature). Dimethoxytriphenylmethyl chloride (1765.7 g,5.21 mol) was added as a solid in one portion. The reaction was allowedto warm to −2° C. over 1 h. (Note: The reaction was monitored closely byTLC (EtOAc) to determine when to stop the reaction so as to not generatethe undesired bis-DMT substituted side product). The reaction wasallowed to warm from −2 to 3° C. over 25 min. then quenched by addingMeOH (300 mL) followed after 10 min by toluene (16 L) and water (16 L).The solution was transferred to a clear 50 L vessel with a bottomoutlet, vigorously stirred for 1 minute, and the layers separated. Theaqueous layer was removed and the organic layer was washed successivelywith 10% aqueous citric acid (8 L) and water (12 L). The product wasthen extracted into the aqueous phase by washing the toluene solutionwith aqueous sodium hydroxide (0.5N, 16 L and 8 L). The combined aqueouslayer was overlayed with toluene (12 L) and solid citric acid (8 moles,1270 g) was added with vigorous stirring to lower the pH of the aqueouslayer to 5.5 and extract the product into the toluene. The organic layerwas washed with water (10 L) and TLC of the organic layer indicated atrace of DMT-O-Me, bis DMT and dimer DMT.

[0170] The toluene solution was applied to a silica gel column (6 Lsintered glass funnel containing approx. 2 kg of silica gel slurriedwith toluene (2 L) and TEA(25 mL)) and the fractions were eluted withtoluene (12 L) and EtOAc (3×4 L) using vacuum applied to a filter flaskplaced below the column. The first EtOAc fraction containing both thedesired product and impurities were resubjected to column chromatographyas above. The clean fractions were combined, rotary evaporated to afoam, coevaporated with acetonitrile (6 L) and dried in a vacuum oven(0.1 mm Hg, 40 h, 40° C.) to afford 2850 g of a white crisp foam. NMRspectroscopy indicated a 0.25 mole % remainder of acetonitrile(calculates to be approx. 47 g) to give a true dry weight of 2803 g(96%). HPLC indicated that the product was 99.41% pure, with theremainder being 0.06 DMT-O-Me, 0.10 unknown, 0.44 bis DMT, and nodetectable dimer DMT or 3′-O-DMT.

[0171] Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-0-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite)

[0172]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridine(1237 g, 2.0 mol) was dissolved in anhydrous DMF (2.5 L). The solutionwas co-evaporated with toluene (200 ml) at 50° C. under reducedpressure, then cooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (70 g,1.0 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (20 ml) was added and the solution was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (3.5 L) and water (600 ml) and extracted with hexane(3×3L). The mixture was diluted with water (1.6 L) and extracted withthe mixture of toluene (12 L) and hexanes (9 L). The upper layer waswashed with DMF-water (7:3 v/v, 3×3 L) and water (3×3 L). The organiclayer was dried (Na₂SO₄), filtered and evaporated. The residue wasco-evaporated with acetonitrile (2×2 L) under reduced pressure and driedin a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1526 g of anoff-white foamy solid (95%).

[0173] Preparation of5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate

[0174] To a 50 L Schott glass-lined steel reactor equipped with anelectric stirrer, reagent addition pump (connected to an additionfunnel), heating/cooling system, internal thermometer and argon gas linewas added 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-uridine(2.616 kg, 4.23 mol, purified by base extraction only and no scrubcolumn), anhydrous acetonitrile (20 L), and TEA (9.5 L, 67.7 mol, 16eq). The mixture was chilled with stirring to −10° C. internaltemperature (external −20° C.). Trimethylsilylchloride (1.60 L, 12.7mol, 3.0 eq) was added over 30 min. while maintaining the internaltemperature below −5° C., followed by a wash of anhydrous acetonitrile(1 L). (Note: the reaction is mildly exothermic and copious hydrochloricacid fumes form over the course of the addition). The reaction wasallowed to warm to 0° C. and the reaction progress was confirmed by TLC(EtOAc, R_(f) 0.68 and 0.87 for starting material and silyl product,respectively). Upon completion, triazole (2.34 kg, 33.8 mol, 8.0 eq) wasadded the reaction was cooled to −20° C. internal temperature (external−30° C.). Phosphorous oxychloride (793 mL, 8.51 mol, 2.01 eq) was addedslowly over 60 min so as to maintain the temperature between −20° C. and−10° C. (note: strongly exothermic), followed by a wash of anhydrousacetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1h, at which point it was an off-white thick suspension. TLC indicated acomplete conversion to the triazole product (EtOAc, R_(f) 0.87 to 0.75with the product spot glowing in long wavelength UV light). The reactionwas cooled to −15° C. and water (5 L) was slowly added at a rate tomaintain the temperature below +10° C. in order to quench the reactionand to form a homogenous solution. (Caution: this reaction is initiallyvery strongly exothermic). Approximately one-half of the reaction volume(22 L) was transferred by air pump to another vessel, diluted with EtOAc(12 L) and extracted with water (2×8 L). The second half of the reactionwas treated in the same way. The combined aqueous layers wereback-extracted with EtOAc (8 L) The organic layers were combined andconcentrated in a 20 L rotary evaporator to an oily foam. The foam wascoevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note:dioxane may be used instead of anhydrous acetonitrile if dried to a hardfoam). The residue was dissolved in dioxane (2 L) and concentratedammonium hydroxide (750 mL) was added. A homogenous solution formed in afew minutes and the reaction was allowed to stand overnight

[0175] TLC indicated a complete reaction (CH₂Cl₂-acetone-MeOH, 20:5:3,R_(f) 0.51). The reaction solution was concentrated on a rotaryevaporator to a dense foam and slowly redissolved in warm CH₂Cl₂ (4 L,40° C.) and transferred to a 20 L glass extraction vessel equipped witha air-powered stirrer. The organic layer was extracted with water (2×6L) to remove the triazole by-product. (Note: In the first extraction anemulsion formed which took about 2 h to resolve). The water layer wasback-extracted with CH₂Cl₂ (2×2 L), which in turn was washed with water(3 L). The combined organic layer was concentrated in 2×20 L flasks to agum and then recrystallized from EtOAc seeded with crystalline product.After sitting overnight, the first crop was collected on a 25 cm CoorsBuchner funnel and washed repeatedly with EtOAc until a whitefree-flowing powder was left (about 3×3 L). The filtrate wasconcentrated to an oil recrystallized from EtOAc, and collected asabove. The solid was air-dried in pans for 48 h, then further dried in avacuum oven (50° C., 0.1 mm Hg, 17 h) to afford 2248 g of a brightwhite, dense solid (86%). An HPLC analysis indicated both crops to be99.4% pure and NMR spectroscopy indicated only a faint trace of EtOAcremained.

[0176] Preparation of5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methyl-cytidinepenultimate intermediate:

[0177] Crystalline5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-cytidine (1000 g,1.62 mol) was suspended in anhydrous DMF (3 kg) at ambient temperatureand stirred under an Ar atmosphere. Benzoic anhydride (439.3 g, 1.94mol) was added in one portion. The solution clarified after 5 hours andwas stirred for 16 h. HPLC indicated 0.45% starting material remained(as well as 0.32% N4, 3′-O-bis Benzoyl). An additional amount of benzoicanhydride (6.0 g, 0.0265 mol) was added and after 17 h, HPLC indicatedno starting material was present. TEA (450 mL, 3.24 mol) and toluene (6L) were added with stirring for 1 minute. The solution was washed withwater (4×4 L), and brine (2×4 L). The organic layer was partiallyevaporated on a 20 L rotary evaporator to remove 4 L of toluene andtraces of water. HPLC indicated that the bis benzoyl side product waspresent as a 6% impurity. The residue was diluted with toluene (7 L) andanhydrous DMSO (200 mL, 2.82 mol) and sodium hydride (60% in oil, 70 g,1.75 mol) was added in one portion with stirring at ambient temperatureover 1 h. The reaction was quenched by slowly adding then washing withaqueous citric acid (10%, 100 mL over 10 min, then 2×4 L), followed byaqueous sodium bicarbonate (2%, 2 L), water (2×4 L) and brine (4 L). Theorganic layer was concentrated on a 20 L rotary evaporator to about 2 Ltotal volume. The residue was purified by silica gel columnchromatography (6 L Buchner funnel containing 1.5 kg of silica gelwetted with a solution of EtOAc-hexanes-TEA(70:29:1)). The product waseluted with the same solvent (30 L) followed by straight EtOAc (6 L).The fractions containing the product were combined, concentrated on arotary evaporator to a foam and then dried in a vacuum oven (50° C., 0.2mm Hg, 8 h) to afford 1155 g of a crisp, white foam (98%). HPLCindicated a purity of >99.7%.

[0178] Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite)

[0179]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidine(1082 g, 1.5 mol) was dissolved in anhydrous DMF (2 L) and co-evaporatedwith toluene (300 ml) at 50° C. under reduced pressure. The mixture wascooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5g, 0.75 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (30 ml) was added, and the mixture was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (1 L) and water (400 ml) and extracted with hexane (3×3L). The mixture was diluted with water (1.2 L) and extracted with amixture of toluene (9 L) and hexanes (6 L). The two layers wereseparated and the upper layer was washed with DMF-water (60:40 v/v, 3×3L) and water (3×2 L). The organic layer was dried (Na₂SO₄), filtered andevaporated. The residue was co-evaporated with acetonitrile (2×2 L)under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40h) to afford 1336 g of an off-white foam (97%).

[0180] Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite)

[0181]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosine(purchased from Reliable Biopharmaceutical, St. Lois, Mo.), 1098 g, 1.5mol) was dissolved in anhydrous DMF (3 L) and co-evaporated with toluene(300 ml) at 50° C. The mixture was cooled to room temperature and2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) andtetrazole (78.8 g, 1.24 mol) were added. The mixture was shaken untilall tetrazole was dissolved, N-methylimidazole (30 ml) was added, andmixture was left at room temperature for 5 hours. TEA (300 ml) wasadded, the mixture was diluted with DMF (1 L) and water (400 ml) andextracted with hexanes (3×3 L). The mixture was diluted with water (1.4L) and extracted with the mixture of toluene (9 L) and hexanes (6 L).The two layers were separated and the upper layer was washed withDMF-water (60:40, v/v, 3×3 L) and water (3×2 L). The organic layer wasdried (Na₂SO₄), filtered and evaporated to a sticky foam. The residuewas co-evaporated with acetonitrile (2.5 L) under reduced pressure anddried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1350 g of anoff-white foam solid (96%).

[0182] Prepartion of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite)

[0183]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrlguanosine(purchased from Reliable Biopharmaceutical, St. Louis, Mo., 1426 g, 2.0mol) was dissolved in anhydrous DMF (2 L). The solution wasco-evaporated with toluene (200 ml) at 50° C., cooled to roomtemperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g,3.0 mol) and tetrazole (68 g, 0.97 mol) were added. The mixture wasshaken until all tetrazole was dissolved, N-methylimidazole (30 ml) wasadded, and the mixture was left at room temperature for 5 hours. TEA(300 ml) was added, the mixture was diluted with DMF (2 L) and water(600 ml) and extracted with hexanes (3×3 L). The mixture was dilutedwith water (2 L) and extracted with a mixture of toluene (10 L) andhexanes (5 L). The two layers were separated and the upper layer waswashed with DMF-water (60:40, v/v, 3×3 L). EtOAc (4 L) was added and thesolution was washed with water (3×4 L). The organic layer was dried(Na₂SO₄), filtered and evaporated to approx. 4 kg. Hexane (4 L) wasadded, the mixture was shaken for 10 min, and the supernatant liquid wasdecanted. The residue was co-evaporated with acetonitrile (2×2 L) underreduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) toafford 1660 g of an off-white foamy solid (91%).

[0184] 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites

[0185] 2′-(Dimethylaminooxyethoxy) nucleoside amidites

[0186] 2′-(Dimethylaminooxyethoxy) nucleoside amidites (also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites) areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

[0187] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0188] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (R_(f) 0.22, EtOAc) indicated a complete reaction. Thesolution was concentrated under reduced pressure to a thick oil. Thiswas partitioned between CH₂Cl₂ (1 L) and saturated sodium bicarbonate(2×1 L) and brine (1 L). The organic layer was dried over sodiumsulfate, filtered, and concentrated under reduced pressure to a thickoil. The oil was dissolved in a 1:1 mixture of EtOAc and ethyl ether(600 mL) and cooling the solution to −10° C. afforded a whitecrystalline solid which was collected by filtration, washed with ethylether (3×2 00 mL) and dried (40° C., 1 mm Hg, 24 h) to afford 149 g ofwhite solid (74.8%). TLC and NMR spectroscopy were consistent with pureproduct.

[0189]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0190] In the fume hood, ethylene glycol (350 mL, excess) was addedcautiously with manual stirring to a 2 L stainless steel pressurereactor containing borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL).(Caution : evolves hydrogen gas).5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure <100 psig). The reaction vessel was cooled to ambienttemperature and opened. TLC (EtOAc, R_(f) 0.67 for desired product andR_(f) 0.82 for ara-T side product) indicated about 70% conversion to theproduct. The solution was concentrated under reduced pressure (10 to 1mm Hg) in a warm water bath (40-100° C.) with the more extremeconditions used to remove the ethylene glycol. (Alternatively, once theTHF has evaporated the solution can be diluted with water and theproduct extracted into EtOAc). The residue was purified by columnchromatography (2 kg silica gel, EtOAc-hexanes gradient 1:1 to 4:1). Theappropriate fractions were combined, evaporated and dried to afford 84 gof a white crisp foam (50%), contaminated starting material (17.4 g, 12%recovery) and pure reusable starting material (20 g, 13% recovery). TLCand NMR spectroscopy were consistent with 99% pure product.

[0191]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0192]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol) and dried over P₂O₅ underhigh vacuum for two days at 40° C. The reaction mixture was flushed withargon and dissolved in dry THF (369.8 mL, Aldrich, sure seal bottle).Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to thereaction mixture with the rate of addition maintained such that theresulting deep red coloration is just discharged before adding the nextdrop. The reaction mixture was stirred for 4 hrs., after which time TLC(EtOAc:hexane, 60:40) indicated that the reaction was complete. Thesolvent was evaporated in vacuuo and the residue purified by flashcolumn chromatography (eluted with 60:40 EtOAc:hexane), to yield2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%) upon rotary evaporation.

[0193]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0194]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate washed with ice coldCH₂Cl₂, and the combined organic phase was washed with water and brineand dried (anhydrous Na₂SO₄). The solution was filtered and evaporatedto afford 2′-O-(aminooxyethyl) thymidine, which was then dissolved inMeOH (67.5 mL). Formaldehyde (20% aqueous solution, w/w, 1.1 eq.) wasadded and the resulting mixture was stirred for 1 h. The solvent wasremoved under vacuum and the residue was purified by columnchromatography to yield5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridineas white foam (1.95 g, 78%) upon rotary evaporation.

[0195] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,Ndimethylaminooxyethyl]-5-methyluridine

[0196]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL) and cooled to 10° C.under inert atmosphere. Sodium cyanoborohydride (0.39 g, 6.13 mmol) wasadded and the reaction mixture was stirred. After 10 minutes thereaction was warmed to room temperature and stirred for 2 h. while theprogress of the reaction was monitored by TLC (5% MeOH in CH₂Cl₂).Aqueous NaHCO₃ solution (5%, 10 mL) was added and the product wasextracted with EtOAc (2×20 mL). The organic phase was dried overanhydrous Na₂SO₄, filtered, and evaporated to dryness. This entireprocedure was repeated with the resulting residue, with the exceptionthat formaldehyde (20% w/w, 30 mL, 3.37 mol) was added upon dissolutionof the residue in the PPTS/MeOH solution. After the extraction andevaporation, the residue was purified by flash column chromatography and(eluted with 5% MeOH in CH₂Cl₂) to afford5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%) upon rotary evaporation.

[0197] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0198] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and TEA (1.67 mL, 12 mmol, dry, stored over KOH) and added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol). The reaction was stirred at room temperature for 24hrs and monitored by TLC (5% MeOH in CH₂Cl₂). The solvent was removedunder vacuum and the residue purified by flash column chromatography(eluted with 10% MeOH in CH₂Cl₂) to afford2′-O-(dimethylaminooxyethyl)-5-methyluridine (766mg, 92.5%) upon rotaryevaporation of the solvent.

[0199] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0200] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C., co-evaporatedwith anhydrous pyridine (20 mL), and dissolved in pyridine (11 mL) underargon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol) and4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) were added to thepyridine solution and the reaction mixture was stirred at roomtemperature until all of the starting material had reacted. Pyridine wasremoved under vacuum and the residue was purified by columnchromatography (eluted with 10% MeOH in CH₂Cl₂ containing a few drops ofpyridine) to yield5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%)upon rotary evaporation.

[0201]5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0202] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL), N,N-diisopropylaminetetrazonide (0.29 g, 1.67 mmol) was added and the mixture was dried overP₂O₅ under high vacuum overnight at 40° C. This was dissolved inanhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 h under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:EtOAc 1:1). The solvent was evaporated, thenthe residue was dissolved in EtOAc (70 mL) and washed with 5% aqueousNaHCO₃ (40 mL). The EtOAc layer was dried over anhydrous Na₂SO₄,filtered, and concentrated. The residue obtained was purified by columnchromatography (EtOAc as eluent) to afford5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%) upon rotary evaporation.

[0203] 2′-(Aminooxyethoxy) nucleoside amidites

[0204] 2′-(Aminooxyethoxy) nucleoside amidites (also known in the art as2′-O-(aminooxyethyl) nucleoside amidites) are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

[0205]N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0206] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside may bephosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0207] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

[0208] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known inthe art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂,or 2′-DMAEOE nucleoside amidites) are prepared as follows. Othernucleoside amidites are prepared similarly.

[0209] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

[0210] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) wasslowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10mmol) with stirring in a 100 mL bomb. (Caution: Hydrogen gas evolves asthe solid dissolves). O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol),and sodium bicarbonate (2.5 mg) were added and the bomb was sealed,placed in an oil bath and heated to 155° C. for 26 h. then cooled toroom temperature. The crude solution was concentrated, the residue wasdiluted with water (200 mL) and extracted with hexanes (200 mL). Theproduct was extracted from the aqueous layer with EtOAc (3×200 mL) andthe combined organic layers were washed once with water, dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by silica gel column chromatography (eluted with 5:100:2MeOH/CH₂Cl₂/TEA) as the eluent. The appropriate fractions were combinedand evaporated to afford the product as a white solid.

[0211] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine

[0212] To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), was added TEA (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) and the reaction was stirred for 1 h. Thereaction mixture was poured into water (200 mL) and extracted withCH₂Cl₂ (2×200 mL). The combined CH₂Cl₂ layers were washed with saturatedNaHCO₃ solution, followed by saturated NaCl solution, dried overanhydrous sodium sulfate, filtered and evaporated. The residue waspurified by silica gel column chromatography (eluted with 5:100:1MeOH/CH₂Cl₂/TEA) to afford the product.

[0213]5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0214] Diisopropylaminotetrazolide (0.6 g) and2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) were addedto a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture was stirred overnight and the solventevaporated. The resulting residue was purified by silica gel columnchromatography with EtOAc as the eluent to afford the title compound.

Example 2

[0215] Oligonucleotide Synthesis

[0216] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

[0217] Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

[0218] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0219] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0220] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0221] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0222] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0223] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0224] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3

[0225] Oligonucleoside Synthesis

[0226] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedimethyl-hydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0227] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0228] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0229] PNA Synthesis

[0230] Peptide nucleic acids (PNAs) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

[0231] Synthesis of Chimeric Oligonucleotides

[0232] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0233] [2′-O-Me]—[2′-deoxy]—[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

[0234] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[0235] [2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[0236] [2′-O-(2-methoxyethyl)]—[2′-deoxy]—[-2′-O-(methoxyethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites. [2′-O-(2-Methoxyethyl)Phosphodiester]—[2′-deoxyPhosphorothioate]—[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[0237] [2′-O-(2-methoxyethyl phosphodiester]—[2′-deoxyphosphorothioate]—[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0238] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0239] Oligonucleotide Isolation

[0240] After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

[0241] Oligonucleotide Synthesis—96 Well Plate Format

[0242] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a 96-well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

[0243] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

[0244] Oligonucleotide Analysis—96-Well Plate Format

[0245] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

[0246] Cell Culture and Oligonucleotide Treatment

[0247] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,ribonuclease protection assays, or RT-PCR.

[0248] T-24 Cells:

[0249] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

[0250] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0251] A549 Cells:

[0252] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

[0253] NHDF Cells:

[0254] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville, Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville, Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

[0255] HEK Cells:

[0256] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville, Md.). HEKs were routinelymaintained in Keratinocyte Growth Medium (Clonetics Corporation,Walkersville, Md.) formulated as recommended by the supplier. Cells wereroutinely maintained for up to 10 passages as recommended by thesupplier.

[0257] Treatment with Antisense Compounds:

[0258] When cells reached 70% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.After 4-7 hours of treatment, the medium was replaced with fresh medium.Cells were harvested 16-24 hours after oligonucleotide treatment.

[0259] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770)mRNA is then utilized as the screening concentration for newoligonucleotides in subsequent experiments for that cell line. If 80%inhibition is not achieved, the lowest concentration of positive controloligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA isthen utilized as the oligonucleotide screening concentration insubsequent experiments for that cell line. If 60% inhibition is notachieved, that particular cell line is deemed as unsuitable foroligonucleotide transfection experiments. The concentrations ofantisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 10

[0260] Analysis of oligonucleotide inhibition of PPP2R1A expression

[0261] Antisense modulation of PPP2R1A expression can be assayed in avariety of ways known in the art. For example, PPP2R1A mRNA levels canbe quantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+mRNA. The preferred method of RNA analysis ofthe present invention is the use of total cellular RNA as described inother examples herein. Methods of RNA isolation are taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993. Northern blot analysis is routine in the art and is taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions.

[0262] Protein levels of PPP2R1A can be quantitated in a variety of wayswell known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), ELISA or fluorescence-activated cell sorting(FACS). Antibodies directed to PPP2R1A can be identified and obtainedfrom a variety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., (CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997). Preparation of monoclonal antibodies istaught in, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 2, pp 11.4.1-11.11.5, John Wiley & Sons, Inc.,1997).

[0263] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons,Inc., 1998). Western blot (immunoblot) analysis is standard in the artand can be found at, for example, Ausubel, F. M. et al., (CurrentProtocols in Molecular Biology, Volume 2, pp 10.8.1-10.8.21, John Wiley& Sons, Inc., 1997). Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., (Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley & Sons, Inc., 1991).

Example 11

[0264] Poly(A)+mRNA Isolation

[0265] Poly(A)+mRNA was isolated according to Miura et al., (Clin.Chem., 1996, 42, 1758-1764). Other methods for poly(A)+mRNA isolationare taught in, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993). Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70° C., was added to each well, the plate wasincubated on a 90° C. hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

[0266] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12

[0267] Total RNA Isolation

[0268] Total RNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia, Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 150 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 150 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a wastecollection tray and attached to a vacuum source. Vacuum was applied for1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and incubated for 15 minutes and the vacuum was again applied for1 minute. An additional 500 μL of Buffer RW1 was added to each well ofthe RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL ofBuffer RPE was then added to each well of the RNEASY 96™ plate and thevacuum applied for a period of 90 seconds. The Buffer RPE wash was thenrepeated and the vacuum was applied for an additional 3 minutes. Theplate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 170 μL water into each well, incubating1 minute, and then applying the vacuum for 3 minutes.

[0269] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

[0270] Real-time Quantitative PCR Analysis of PPP2R1A mRNA Levels

[0271] Quantitation of PPP2R1A mRNA levels was determined by real-timequantitative PCR using the ABI PRISM™ 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCRin which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems,Foster City, Calif., Operon Technologies Inc., Alameda, Calif. orIntegrated DNA Technologies Inc., Coralville, Iowa) is attached to the5′ end of the probe and a quencher dye (e.g., TAMRA, obtained fromeither PE-Applied Biosystems, Foster City, Calif., Operon TechnologiesInc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville,Iowa) is attached to the 3′ end of the probe. When the probe and dyesare intact, reporter dye emission is quenched by the proximity of the 3′quencher dye. During amplification, annealing of the probe to the targetsequence creates a substrate that can be cleaved by the 5′-exonucleaseactivity of Taq polymerase. During the extension phase of the PCRamplification cycle, cleavage of the probe by Taq polymerase releasesthe reporter dye from the remainder of the probe (and hence from thequencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™7700 Sequence Detection System. In each assay, a series of parallelreactions containing serial dilutions of mRNA from untreated controlsamples generates a standard curve that is used to quantitate thepercent inhibition after antisense oligonucleotide treatment of testsamples.

[0272] Prior to quantitative PCR analysis, primer-probe sets specific tothe target gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

[0273] PCR reagents were obtained from Invitrogen Corporation,(Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μLPCR cocktail (2.5×PCR buffer (-MgCl2), 6.6 mM MgCl2, 375 μM each ofDATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverseprimer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM®Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-wellplates containing 30 μL total RNA solution. The RT reaction was carriedout by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0274] Gene target quantities obtained by real time RT-PCR arenormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreen™(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent from Molecular Probes. Methods of RNAquantification by RiboGreen™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

[0275] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipettedinto a 96-well plate containing 30 μL purified, cellular RNA. The plateis read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at480 nm and emission at 520 nm.

[0276] Probes and primers to human PPP2R1A were designed to hybridize toa human PPP2R1A sequence, using published sequence information (GenBankaccession number J02902.1, incorporated herein as SEQ ID NO:4). Forhuman PPP2R1A the PCR primers were: forward primer:CACCGCATGACTACGCTCTTC (SEQ ID NO: 5) reverse primer:GCATGTGCTTGGTGGTGATG (SEQ ID NO: 6) and the PCR probe was:FAM-TGTGCTGTCTGAGGTCTGTGGGCA-TAMRA (SEQ ID NO: 7) where FAM is thefluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCRprimers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8) reverseprimer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is thefluorescent reporter dye and TAMRA is the quencher dye.

Example 14

[0277] Northern Blot Analysis of PPP2R1A mRNA Levels

[0278] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0279] To detect human PPP2R1A, a human PPP2R1A specific probe wasprepared by PCR using the forward primer CACCGCATGACTACGCTCTTC (SEQ IDNO: 5) and the reverse primer GCATGTGCTTGGTGGTGATG (SEQ ID NO: 6). Tonormalize for variations in loading and transfer efficiency membraneswere stripped and probed for human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0280] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15

[0281] Antisense Inhibition of Human PPP2R1A Expression by ChimericPhosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap

[0282] In accordance with the present invention, a series ofoligonucleotides were designed to target different regions of the humanPPP2R1A RNA, using published sequences (GenBank accession numberJ02902.1, incorporated herein as SEQ ID NO: 4, residues 1666448-1705625of GenBank accession number NT_(—)011091.5, representing a genomicsequence of PPP2R1A, incorporated herein as SEQ ID NO: 11, and GenBankaccession number AA903754.1, the complement of which is incorporatedherein as SEQ ID NO: 12). The oligonucleotides are shown in Table 1.“Target site” indicates the first (5′-most) nucleotide number on theparticular target sequence to which the olgonucleotide binds. Allcompounds in Table 1 are chemeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human PPP2R1A mRNA levels by quantitative real-time PCRas described in other examples herein. Data are averages from twoexperiments in which T-24 cells were treated with the oligonucleotidesof the present invention. The positive control for each datapoint isidentified in the table by sequence ID number. If present, “N.D.”indicates “no data”. TABLE 1 Inhibition of human PPP2R1A mRNA levels bychimeric phosphorothioate oligonucleotides having 2′-MOE wings and adeoxy gap TARGET CONTROL SEQ ID TARGET SEQ ID SEQ ID ISIS # REGION NOSITE SEQUENCE % INHIB NO NO 155001 Coding 4 1736 gcggacattggcaaccgggt 413 2 155002 Coding 4 1095 acatgatcacattctcccga 3 14 2 155003 Coding 41729 ttggcaaccgggtccccagc 68 15 2 155004 Coding 4 1544aaacttttccactagcttct 50 16 2 155005 Coding 4 1097 ggacatgatcacattctccc80 17 2 155006 Coding 4 1064 gaggttttcacagaactctt 54 18 2 155007 Coding4 645 acaggttccggaagtactgt 58 19 2 155008 Coding 4 877gcctggcgcagagtgggcat 28 20 2 155009 Coding 4 1724 aaccgggtccccagccatgc82 21 2 155010 Stop 4 1895 tcaggcgagagacagaacag 63 22 2 Codon 155011Coding 4 1636 cagaagagcgtagtcatgcg 20 23 2 155012 3′UTR 4 2008tcagaccatgcacagggagt 27 24 2 155013 Coding 4 1430 tcccagctgtccagccagga48 25 2 155014 Coding 4 1475 ccaggccatgcacaaggagt 78 26 2 155015 Coding4 1831 tgggtcagcttctctaggat 10 27 2 155016 Coding 4 1286gttagagatgatgttcagcc 70 28 2 155017 Coding 4 344 cagctgttctgccagggcca 4629 2 155018 Coding 4 905 gcggacggcccaggacttgt 0 30 2 155019 Coding 4 390ggcagtgcacgtactctggg 54 31 2 155020 Coding 4 990 tctggaaggcagggaccagg 5232 2 155021 Coding 4 1573 atgattgtggcatgggccca 56 33 2 155022 Coding 41180 agacccatgatgactgaggc 80 34 2 155023 Coding 4 1100ctgggacatgatcacattct 75 35 2 155024 Coding 4 978 ggaccaggtctgtcttggtg 6036 2 155025 5′UTR 4 113 cctttcctgtcagactgcgg 72 37 2 155026 Coding 41884 acagaacagtcagagcctcc 36 38 2 155027 Coding 4 638ccggaagtactgtcgaagtt 49 39 2 155028 Coding 4 1885 gacagaacagtcagagcctc45 40 2 155029 Coding 4 577 aagaggccgcaggccgaggt 52 41 2 155030 Coding 4768 cagaggccaggttggagaac 45 42 2 155031 Coding 4 1094catgatcacattctcccgac 79 43 2 155032 Coding 4 1664 cccacagacctcagacagca51 44 2 155033 3′UTR 4 1918 cagtgtttgctcctcttcca 91 45 2 155034 Coding 4980 agggaccaggtctgtcttgg 12 46 2 155035 Coding 4 1107gcaagatctgggacatgatc 78 47 2 155036 Coding 4 1142 ttggttggcatcggacacca66 48 2 155037 5′UTR 4 73 actcccccatggagcggcgg 8 49 2 209374 5′UTR 4 29tgccaaggtgctggagctgg 0 50 2 209376 5′UTR 4 55 gggccggccgtccaagctgg 0 512 209378 Start 4 137 ggccgccgccatcttggctc 68 52 2 Codon 209380 Coding 4185 gagttcgtctatgagcaccg 79 53 2 209382 Coding 4 230cagcttcttgatgctgttga 90 54 2 209384 Coding 4 286 aaaggcagaagctcacttcg 7355 2 209386 Coding 4 361 agggtagtgaaggttcccag 76 56 2 209388 Coding 4404 cagcggtggcagcaggcagt 60 57 2 209390 Coding 4 479ctcgtgtgagatggcccgta 96 58 2 209392 Coding 4 555 gggaggtgaaccagtcgccg 5359 2 209394 Coding 4 615 ccttcacagcactggacact 77 60 2 209396 Coding 4705 tggcaaactcccccagcttg 82 61 2 209398 Coding 4 786gcaccgagtcctgctcgtca 80 62 2 209400 Coding 4 1049 ctctttgaccttgtgggagg96 63 2 209402 Coding 4 1128 acaccagctccttgatgcag 66 64 2 209404 Coding4 1161 ccagggcagacttgacatgt 82 65 2 209406 Coding 4 1219aggtgctcgatggtgttgtc 65 66 2 209408 Coding 4 1306 acctcgttcacacagtccag91 67 2 209410 Coding 4 1461 aggagttaagtttctcatca 79 68 2 209412 Coding4 1691 tagcatgtgcttggtggtga 95 69 2 209414 Coding 4 1796ctgcaaggtgctgttgtcca 73 70 2 209416 Stop 4 1905 tcttccagcatcaggcgaga 7771 2 Codon 209418 3′UTR 4 2067 gtgagacatcttcccaggct 95 72 2 209420 3′UTR4 2182 tcggtgaattgccacctccg 0 73 2 209422 Intron 11 1998gcggccctcccctacttgga 0 74 2 209424 Intron 11 9711 tgcttgagaaagatggccta77 75 2 209426 Exon: 11 13474 cctttgttacctgtaaggaa 0 76 2 IntronJunction 209428 Intron 11 14558 ggatcatcactaggtccaag 80 77 2 209431Exon: 11 22932 agagactcactgtcgaagtt 48 78 2 Intron Junction 209432Intron 11 26841 taaagatctgttaccagaag 24 79 2 209434 Intron: 11 27218ctttctggagctgcagacag 34 80 2 Exon Junction 209437 Exon: 11 28103gtgcccttacctcatccttc 11 81 2 Intron Junction 209438 3′UTR 12 524tcccattacagcagcaggat 98 82 2 209441 3′UTR 12 572 accaatgatgagaaggacgg 3683 2 209443 3′UTR 12 648 gacctttatttctctgtgaa 88 84 2

[0283] As shown in Table 1, SEQ ID NOs 15, 17, 21, 22, 26, 28, 34, 35,36, 37, 43, 45, 47, 48, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 75, 77, 82 and 84 demonstrated at least60% inhibition of human PPP2R1A expression in this assay and aretherefore preferred. The target sites to which these preferred sequencesare complementary are herein referred to as “preferred target regions”and are therefore preferred sites for targeting by compounds of thepresent invention. These preferred target regions are shown in Table 2.The sequences represent the reverse complement of the preferredantisense compounds shown in Table 1. “Target site” indicates the first(5′-most) nucleotide number of the corresponding target nucleic acid.Also shown in Table 2 is the species in which each of the preferredtarget regions was found. TABLE 2 Sequence and position of preferredtarget regions identified in PPP2R1A. TARGET SEQ ID TARGET REV COMP SEQID SITEID NO SITE SEQUENCE OF SEQ ID ACTIVE IN NO 70548 4 1729gctggggacccggttgccaa 15 H. sapiens 85 70550 4 1097 gggagaatgtgatcatgtcc17 H. sapiens 86 70554 4 1724 gcatggctggggacccggtt 21 H. sapiens 8770555 4 1895 ctgttctgtctctcgcctga 22 H. sapiens 88 70559 4 1475actccttgtgcatggcctgg 26 H. sapiens 89 70561 4 1286 ggctgaacatcatctctaac28 H. sapiens 90 70567 4 1180 gcctcagtcatcatgggtct 34 H. sapiens 9170568 4 1100 agaatgtgatcatgtcccag 35 H. sapiens 92 70569 4 978caccaagacagacctggtcc 36 H. sapiens 93 70570 4 113 ccgcagtctgacaggaaagg37 H. sapiens 94 70576 4 1094 gtcgggagaatgtgatcatg 43 H. sapiens 9570578 4 1918 tggaagaggagcaaacactg 45 H. sapiens 96 70580 4 1107gatcatgtcccagatcttgc 47 H. sapiens 97 70581 4 1142 tggtgtccgatgccaaccaa48 H. sapiens 98 127017 4 137 gagccaagatggcggcggcc 52 H. sapiens 99127018 4 185 cggtgctcatagacgaactc 53 H. sapiens 100 127019 4 230tcaacagcatcaagaagctg 54 H. sapiens 101 127020 4 286 cgaagtgagcttctgccttt55 H. sapiens 102 127021 4 361 ctgggaaccttcactaccct 56 H. sapiens 103127022 4 404 actgcctgctgccaccgctg 57 H. sapiens 104 127023 4 479tacgggccatctcacacgag 58 H. sapiens 105 127025 4 615 agtgtccagtgctgtgaagg60 H. sapiens 106 127026 4 705 caagctgggggagtttgcca 61 H. sapiens 107127027 4 786 tgacgagcaggactcggtgc 62 H. sapiens 108 127028 4 1049cctcccacaaggtcaaagag 63 H. sapiens 109 127029 4 1128ctgcatcaaggagctggtgt 64 H. sapiens 110 127030 4 1161acatgtcaagtctgccctgg 65 H. sapiens 111 127031 4 1219gacaacaccatcgagcacct 66 H. sapiens 112 127032 4 1306ctggactgtgtgaacgaggt 67 H. sapiens 113 127033 4 1461tgatgagaaacttaactcct 68 H. sapiens 114 127034 4 1691tcaccaccaagcacatgcta 69 H. sapiens 115 127035 4 1796tggacaacagcaccttgcag 70 H. sapiens 116 127036 4 1905tctcgcctgatgctggaaga 71 H. sapiens 117 127037 4 2067agcctgggaagatgtctcac 72 H. sapiens 118 127040 11 9711taggccatctttctcaagca 75 H. sapiens 119 127042 11 14558cttggacctagtgatgatcc 77 H. sapiens 120 127047 12 524atcctgctgctgtaatggga 82 H. sapiens 121 127049 12 648ttcacagagaaataaaggtc 84 H. sapiens 122

[0284] As these “preferred target regions” have been found byexperimentation to be open to, and accessible for, hybridzation with theantisense compounds of the present invention, one of skill in the artwill recognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these sites andconsequently inhibit the expression of PPP2R1A.

[0285] In one embodiment, the “preferred target region” may be employedin screening candidate antisense compounds. “Candidate antisensecompounds” are those that inhibit the expression of a nucleic acidmolecule encoding PPP2R1A and which comprise at least an 8-nucleobaseportion which is complementary to a preferred target region. The methodcomprises the steps of contacting a preferred target region of a nucleicacid molecule encoding PPP2R1A with one or more candidate antisensecompounds, and selecting for one or more candidate antisense compoundswhich inhibit the expression of a nucleic acid molecule encodingPPP2R1A. Once it is shown that the candidate antisense compound orcompounds are capable of inhibiting the expression of a nucleic acidmolecule encoding PPP2R1A, the candidate antisense compound may beemployed as an antisense compound in accordance with the presentinvention.

[0286] According to the present invention, antisense compounds includeribozymes, external guide sequence (EGS) oligonucleotides (oligozymes),and other short catalytic RNAs or catalytic oligonucleotides whichhybridize to the target nucleic acid and modulate its expression.

Example 16

[0287] Western Blot Analysis of PPP2R1A Protein Levels

[0288] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to PPP2R1A is used, witha radiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

1 122 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence AntisenseOligonucleotide 2 gtgcgcgcga gcccgaaatc 20 3 20 DNA Artificial SequenceAntisense Oligonucleotide 3 atgcattctg cccccaagga 20 4 2205 DNA H.sapiens CDS (145)...(1914) 4 gaattccggt tctcactctt gacgttgtcc agctccagcaccttggcaac tcccccagct 60 tggacggccg gcccgccgct ccatggggga gtcatctgagcacagctgct ggccgcagtc 120 tgacaggaaa gggacggagc caag atg gcg gcg gcc gacggc gac gac tcg 171 Met Ala Ala Ala Asp Gly Asp Asp Ser 1 5 ctg tac cccatc gcg gtg ctc ata gac gaa ctc cgc aat gag gac gtt 219 Leu Tyr Pro IleAla Val Leu Ile Asp Glu Leu Arg Asn Glu Asp Val 10 15 20 25 cag ctt cgcctc aac agc atc aag aag ctg tcc acc atc gcc ttg gcc 267 Gln Leu Arg LeuAsn Ser Ile Lys Lys Leu Ser Thr Ile Ala Leu Ala 30 35 40 ctt ggg gtt gaaagg acc cga agt gag ctt ctg cct ttc ctt aca gat 315 Leu Gly Val Glu ArgThr Arg Ser Glu Leu Leu Pro Phe Leu Thr Asp 45 50 55 acc atc tat gat gaagat gag gtc ctc ctg gcc ctg gca gaa cag ctg 363 Thr Ile Tyr Asp Glu AspGlu Val Leu Leu Ala Leu Ala Glu Gln Leu 60 65 70 gga acc ttc act acc ctggtg gga ggc cca gag tac gtg cac tgc ctg 411 Gly Thr Phe Thr Thr Leu ValGly Gly Pro Glu Tyr Val His Cys Leu 75 80 85 ctg cca ccg ctg gag tcg ctggcc aca gtg gag gag aca gtg gtg cgg 459 Leu Pro Pro Leu Glu Ser Leu AlaThr Val Glu Glu Thr Val Val Arg 90 95 100 105 gac aag gca gtg gag tcctta cgg gcc atc tca cac gag cac tcg ccc 507 Asp Lys Ala Val Glu Ser LeuArg Ala Ile Ser His Glu His Ser Pro 110 115 120 tct gac ctg gag gcg cacttt gtg ccg cta gtg aag cgg ctg gcg ggc 555 Ser Asp Leu Glu Ala His PheVal Pro Leu Val Lys Arg Leu Ala Gly 125 130 135 ggc gac tgg ttc acc tcccgc acc tcg gcc tgc ggc ctc ttc tcc gtc 603 Gly Asp Trp Phe Thr Ser ArgThr Ser Ala Cys Gly Leu Phe Ser Val 140 145 150 tgc tac ccc cga gtg tccagt gct gtg aag gcg gaa ctt cga cag tac 651 Cys Tyr Pro Arg Val Ser SerAla Val Lys Ala Glu Leu Arg Gln Tyr 155 160 165 ttc cgg aac ctg tgc tcagat gac acc ccc atg gtg cgg cgg gcc gca 699 Phe Arg Asn Leu Cys Ser AspAsp Thr Pro Met Val Arg Arg Ala Ala 170 175 180 185 gcc tcc aag ctg ggggag ttt gcc aag gtg ctg gag ctg gac aac gtc 747 Ala Ser Lys Leu Gly GluPhe Ala Lys Val Leu Glu Leu Asp Asn Val 190 195 200 aag agt gag atc atcccc atg ttc tcc aac ctg gcc tct gac gag cag 795 Lys Ser Glu Ile Ile ProMet Phe Ser Asn Leu Ala Ser Asp Glu Gln 205 210 215 gac tcg gtg cgg ctgctg gcg gtg gag gcg tgc gtg aac atc gcc cag 843 Asp Ser Val Arg Leu LeuAla Val Glu Ala Cys Val Asn Ile Ala Gln 220 225 230 ctt ctg ccc cag gaggat ctg gag gcc ctg gtg atg ccc act ctg cgc 891 Leu Leu Pro Gln Glu AspLeu Glu Ala Leu Val Met Pro Thr Leu Arg 235 240 245 cag gcc gct gaa gacaag tcc tgg gcc gtc cgc tac atg gtg gct gac 939 Gln Ala Ala Glu Asp LysSer Trp Ala Val Arg Tyr Met Val Ala Asp 250 255 260 265 aag ttc aca gagctc cag aaa gca gtg ggg cct gag atc acc aag aca 987 Lys Phe Thr Glu LeuGln Lys Ala Val Gly Pro Glu Ile Thr Lys Thr 270 275 280 gac ctg gtc cctgcc ttc cag aac ctg atg aaa gac tgt gag gcc gag 1035 Asp Leu Val Pro AlaPhe Gln Asn Leu Met Lys Asp Cys Glu Ala Glu 285 290 295 gtg agg gcc gcagcc tcc cac aag gtc aaa gag ttc tgt gaa aac ctc 1083 Val Arg Ala Ala AlaSer His Lys Val Lys Glu Phe Cys Glu Asn Leu 300 305 310 tca gct gac tgtcgg gag aat gtg atc atg tcc cag atc ttg ccc tgc 1131 Ser Ala Asp Cys ArgGlu Asn Val Ile Met Ser Gln Ile Leu Pro Cys 315 320 325 atc aag gag ctggtg tcc gat gcc aac caa cat gtc aag tct gcc ctg 1179 Ile Lys Glu Leu ValSer Asp Ala Asn Gln His Val Lys Ser Ala Leu 330 335 340 345 gcc tca gtcatc atg ggt ctc tct ccc atc ttg ggc aaa gac aac acc 1227 Ala Ser Val IleMet Gly Leu Ser Pro Ile Leu Gly Lys Asp Asn Thr 350 355 360 atc gag cacctc ttg ccc ctc ttc ctg gct cag ctg aag gat gag tgc 1275 Ile Glu His LeuLeu Pro Leu Phe Leu Ala Gln Leu Lys Asp Glu Cys 365 370 375 cct gag gtacgg ctg aac atc atc tct aac ctg gac tgt gtg aac gag 1323 Pro Glu Val ArgLeu Asn Ile Ile Ser Asn Leu Asp Cys Val Asn Glu 380 385 390 gtg att ggcatc cgg cag ctg tcc cag tcc ctg ctc cct gcc att gtg 1371 Val Ile Gly IleArg Gln Leu Ser Gln Ser Leu Leu Pro Ala Ile Val 395 400 405 gag ctg gctgag gac gcc aag tgg cgg gtg cgg ctg gcc atc att gag 1419 Glu Leu Ala GluAsp Ala Lys Trp Arg Val Arg Leu Ala Ile Ile Glu 410 415 420 425 tac atgccc ctc ctg gct gga cag ctg gga gtg gag ttc ttt gat gag 1467 Tyr Met ProLeu Leu Ala Gly Gln Leu Gly Val Glu Phe Phe Asp Glu 430 435 440 aaa cttaac tcc ttg tgc atg gcc tgg ctt gtg gat cat gta tat gcc 1515 Lys Leu AsnSer Leu Cys Met Ala Trp Leu Val Asp His Val Tyr Ala 445 450 455 atc cgcgag gca gcc acc agc aac ctg aag aag cta gtg gaa aag ttt 1563 Ile Arg GluAla Ala Thr Ser Asn Leu Lys Lys Leu Val Glu Lys Phe 460 465 470 ggg aaggag tgg gcc cat gcc aca atc atc ccc aag gtc ttg gcc atg 1611 Gly Lys GluTrp Ala His Ala Thr Ile Ile Pro Lys Val Leu Ala Met 475 480 485 tcc ggagac ccc aac tac ctg cac cgc atg act acg ctc ttc tgc atc 1659 Ser Gly AspPro Asn Tyr Leu His Arg Met Thr Thr Leu Phe Cys Ile 490 495 500 505 aatgtg ctg tct gag gtc tgt ggg cag gac atc acc acc aag cac atg 1707 Asn ValLeu Ser Glu Val Cys Gly Gln Asp Ile Thr Thr Lys His Met 510 515 520 ctaccc acg gtt ctg cgc atg gct ggg gac ccg gtt gcc aat gtc cgc 1755 Leu ProThr Val Leu Arg Met Ala Gly Asp Pro Val Ala Asn Val Arg 525 530 535 ttcaat gtg gcc aag tct ctg cag aag ata ggg ccc atc ctg gac aac 1803 Phe AsnVal Ala Lys Ser Leu Gln Lys Ile Gly Pro Ile Leu Asp Asn 540 545 550 agcacc ttg cag agt gaa gtc aag ccc atc cta gag aag ctg acc cag 1851 Ser ThrLeu Gln Ser Glu Val Lys Pro Ile Leu Glu Lys Leu Thr Gln 555 560 565 gaccag gat gtg gac gtc aaa tac ttt gcc cag gag gct ctg act gtt 1899 Asp GlnAsp Val Asp Val Lys Tyr Phe Ala Gln Glu Ala Leu Thr Val 570 575 580 585ctg tct ctc gcc tga tgctggaaga ggagcaaaca ctggcctctg gtgtccaccc 1954 LeuSer Leu Ala tccaaccccc acaagtccct ctttggggag acactggggg gcctttggctgtcactccct 2014 gtgcatggtc tgaccccagg ccccttcccc cagcacggtt cctcctctccccagcctggg 2074 aagatgtctc actgtccacc tcccaacggc taggggagca cggggttggacaggacagtg 2134 accttgggag gaaggggcta ctccgccatc cttaaaagcc atggagccggaggtggcaat 2194 tcaccgaatt c 2205 5 21 DNA Artificial Sequence PCRPrimer 5 caccgcatga ctacgctctt c 21 6 20 DNA Artificial Sequence PCRPrimer 6 gcatgtgctt ggtggtgatg 20 7 24 DNA Artificial Sequence PCR Probe7 tgtgctgtct gaggtctgtg ggca 24 8 19 DNA Artificial Sequence PCR Primer8 gaaggtgaag gtcggagtc 19 9 20 DNA Artificial Sequence PCR Primer 9gaagatggtg atgggatttc 20 10 20 DNA Artificial Sequence PCR Probe 10caagcttccc gttctcagcc 20 11 39178 DNA H. sapiens 11 ctccgtctcaaaaaaaaaaa aagaaaaaga aaaagaagaa aaaagaaata gatgctgtgg 60 gtattcagatctgtcagagt gctagctcag cctagccctt caagtagcag aataaaggag 120 gaaagagcatcccaggcctt cccatcccca ttcaactccc tcagttagag tggtcctaag 180 attgcggtgggggggtaggg ctgtgttgaa gaggttaaaa acagagatta gtcagatcca 240 atcctgctcccactctatgg aggaaacaaa ttcgaataca tagtcacagc ccaggctctg 300 ggattgagagacagagagag agattggatt ggagagagag agagagagag atgggattgg 360 agagacagagagatagatgg gactggagag acagagagag gggttctgtg gctctggaag 420 cctagagaacagagcctgaa gacagagagg aaagggagta aaggggaggc atttatgcag 480 agatctcagaagtgaatatg aaaggcaccc ctagcagaac gcacagcttg ggcaaggagg 540 cttgctggcatgaaaacgca agaagtgctc aggcagctgt aattctccag gacatcagat 600 aaaaggtaaaaaacagatgg gagacgcgcc tagacagggt attgaatacc agcccgaggg 660 gcttgggtttcatccaaaca gcaaggcaac tgagctcaac acagtgagac tcggtctcca 720 caaacatttttttaaaaatt ggccgggcct agcggcgcgc gccgatagtc ccagctattt 780 ctgcaggctgaggcgagagg atcatttgag ctcggggagg tccaggcggc agtgagccaa 840 gatcctgccactgcactaca gtctgggcaa cagagcgaga ccctgtcttt aaaaaagtaa 900 aaaaaaaaaaaaaaaaaaaa gaaagaaaaa gaaaaaagag agaagggcaa gtcgctccct 960 tctctgggccttggcataaa tcaagcacaa atcaaagtct cactccctcc tcctgccgcg 1020 caacggcgcggagagccagc cagccagcca gcggaaccac ggcctggtaa cccaaaacct 1080 gcacaccctccagctcccca caggacgtca cgtattacca ccgacgcacg cgcagaagcc 1140 ttcccggggactcaagaaag ggcaggctta gcctcctccc catgtcgccc ctcattggct 1200 agaaactactgctcgtctcg gtcgttgtta gcagcgacca gggcgggtac aatcttggtc 1260 gctaggacacggctaacttc cgctttcttc cccctctcct aggctcaaac tagtcaaatc 1320 ttgttcactcgaccaatggc aaatcggaag tgggcgggac ttcacaagtc cggaccaaag 1380 aaacgcgagcttagccctgg gtagcgcggc caatggccgt ggagcagccc ctgtaaactg 1440 gctcgggcgcccccacgccc gcccttcctt cttctcccag cattgccccc cccacgtttc 1500 agcacagcgctggccgcagt ctgacaggaa agggacggag ccaagatggc ggcggccgac 1560 ggcgacgactcgctgtaccc catcgcggtg ctcatagacg aactccgcaa tgaggacgtt 1620 caggtccggaggctacgggg gacttgggga agacgcggag gggtacctgg gggcacgggc 1680 ggccctcgcggagaagactc agcgttcgct gggagtggcg gaagggggcg acggccaatc 1740 agcgtgcgtctcttatctcc ccggttgccc ggactccttg agacggcgct cccgattggg 1800 tgtcggcccagtggagggcg ggggccagcg ctagcctcga gggtcccggg cctgccctgt 1860 gcgcgcggcggtccgcggtc ctgggaggtt gtggccaggg ctggggtctg cggactgggt 1920 ctgggagagaggaggactcc gtgattggcg gcggcctctg aatggcctct tggggatgtg 1980 gggcgcgcatgacttgctcc aagtagggga gggccgccgg gtgggtcggg acctgggaag 2040 gtttttttgttttctgggtt tcgactgctg ggccaagtgg ggaccgagag gcgaaggcct 2100 gccatcctaattcctgctct tcctccgcct ctcattttgg tttaggtgtc ctaagaggac 2160 ggggacgcaaaaacaccccc ccaccaaagg tggggactag ccaagtttag gagcgaatta 2220 ggttgtagaaacccgcctcc ccatctcccc ggatcctccc attgaccagg ataggggttg 2280 agggatttgctaagcagatg aacatttatt tatttctttt ttttgagaca gtctctgtgt 2340 cgcccaggctggggctgggg agcagtggcg cgatctcggc tcagtgcaac ctctgcatac 2400 cgggttcaaaccatcctcgc ccctcagctc cctaattagc tgcgattacc ggcgcgagcc 2460 accacgcccgactaattttt gtatttttag cagagacggg gtttcaccat gttggccagg 2520 cttggtcgggaactcttgac ctcaagcgat ccacccgcct cggcctccca aagtgttgga 2580 attacaggcgtgagccaccg cgcccagccc ggatgaacat tcctggttat gggatgaggt 2640 gacccaaggctctgagccgg gctggtgtgg gattgagaac agttagaact gcaagtccac 2700 ctcccacctgctgtgtgacc ttgtgcaaat tacttcaccg ctttgggtct ctgtgttcca 2760 taaaatatgggctaattgta gtcctggtct tgctggaggg acttgtgagg gactgaacga 2820 attatgacacaaataaaaag tagaatggtg tcaggcctgt tgtaagggct cgataaatat 2880 tagctgtaattattgggagt ggtgattaaa gaggtcccat cctcccttta ggtctgtttt 2940 cccatttataaaattggaca aggtgcactg ggtaacctgc caaacgtggg tctcgcctcc 3000 taggttcctggtatatcact gtttctggtg gcatacaggt tgtggttgta ttcccggccc 3060 ttccacttgttactgattga caagggcaag ttagttaatc tctctaggcc tgtttcctct 3120 tctctttaatggggttaatt acctagttca tagggcggtt gtatgaattc ttttgttcag 3180 taaatatatgccatgtatga gatatgatgc tggggatata gtggagacac cagttgctat 3240 acaggatccagatgtgaaac aggtcagcag agctgaatag atgagtgttt gaaaagtgat 3300 gggaaggaaacaaataggat gtagtggtag cgaatccata ttgaattggg cctctattct 3360 gtgcagggcgctgttctaag cactggtata tagcaggaac aagacagaaa aaactctagg 3420 ccttggggagtttattttgt agtgaggaga gacagacaat aaacagagta tactggggat 3480 aagcattagggagacaaata aaaataagag agatggggta tatatagggc atgcagtttt 3540 aaagtgtggtcaggggaggc cttgttgaga tcctgtttga gaacacgcct gaaagcagag 3600 ggcacagcaagtacaaagtg gggtggggca gatccatttt aagaaggcca atcaggcaag 3660 gcccctctgaggatgcagag tttgagaaga gattgaaaga aggtggggga gagcctccca 3720 ggaagagggaacagatatgt aatggccttt tttttttttt cttctttttt tgagacggag 3780 tcttgttgctctgtttccca ggctggagtg cactgacgcg atctttgctc actgcaacct 3840 ccacctcccgggttcaagca attctcctgc ctcagcctct tgagtagctg ggactacaga 3900 cgcaggccaccacgcctggc tgatttttgt gtgtgtgttt ttagaagaga ctgggtttcc 3960 cggtgttggccaggctggtc tcagactcct gatctcaggt gatcctcctg cctcggcctc 4020 cgaaagtgctggaattacag gcgtgagcca ctatgcccgg ccagtttttt gtttttttta 4080 actccaggatcacttccttc agctatctat cctcccatct tttcattcat ccatccatcc 4140 actaaccagcagacatttgt agggagatgt ctttgtgcca ggatcagagc taggcagctt 4200 accggtttattctaaagaat attgcaaagg atacagatga agacacatgc agggtgaggt 4260 gtgggggaagttgtgtggag cttccatgcc ctccctaggc attccacccc ccaggaaccg 4320 cgaggtgttcatctgtctgg aagctcctat ggctgctttt gtgctacagc ccagagttgc 4380 atggttgtaacagagatggc cgcaaagctt caaatatttg ctgtctaacc ctttgcaggg 4440 aaaaaattgctgacctctga cttaaaccat ctgcgtaaca aattattggg tgcttgctct 4500 atgttaggtacagtacagag gaatatcact gacgatctta ctgaattctc acaatcactc 4560 tttgaagtaggtcctgtcct tgtccacatt ttccagatga ggaaactgaa gcaccataaa 4620 taacatggccaaagctgtgc agctgagaaa tgaaggagcc aggaaaggaa gtaggaccta 4680 agggtggaatagaatctggg gtgggacaca cggttgctga ttatattcat atgccagaca 4740 ttttaattgtgtctgagaca gttcaggata ctagaaggaa catgggacat gaaatcctgt 4800 agtcttgatttatttgttca gcaaatattt atcagcacct cctgtgtgcc agactctgtt 4860 ttagatactagacatacagt agggaacaaa acagcaaagc tgcccttgtg gggtttacat 4920 tctggtttgggagacagata gtagatctgt agtgggatgt cagtggcagt aattgctaag 4980 aagaaaaattgctggacgtg gtggctcaag cctaattgta acactttggt tttattttat 5040 tttattttttttgagacaga gtctcactct gtcgtccagg ccgtctctgc tcattgcaac 5100 ctccgcctcccgggttcaag tgattctcct gcctcagcct cctgagtggc taggattaga 5160 ggcgcccaccaccacatcca gctaattttt gtatttttag tagagagtgg gtttcaccat 5220 gttggccaggctggtctcaa actccctgcc cgcctctgcc tcccaaagtg ctgggattac 5280 aggcatgagccaccacccct ggccaattct agcactttgg aaggccaagg tgaaaggatc 5340 gcttgagcccaggagtttaa gaccagcctg ggcaacaaag tgagaccctg tctctaccaa 5400 aaaaaaaaaaaaaattagct ggtgtgatga cacaggcctt ggtcccagct gctcaggaga 5460 ctgaggtcggaggattgctt gaaccaggga gatcgaggct gcggtgagag cctgggcgac 5520 agcaagaccctgtctccaaa aaaaaaaaaa aaaaaaaaaa agaacaaaat aagacaggta 5580 cagggaatagagcaagcacc tggggctgct ttgttagata gggaggttag agaaagctgc 5640 tctgaggaagtgatgttgaa caaggatttg aatgaaatga acaggtcgtg gaagagcagt 5700 ctgggcagagggaatagttg gtacaaaggc caggagtggg aggatgcttg atgcgttcaa 5760 agcacagcaaaaaggccagt gtgactgagg cagagagaat ggcaaagtgg tgggaggtga 5820 ggactagaccaggactagat caagtgaggc ctgtgggcca aggtgaaggc cttgaagatg 5880 ggagggaggaggaggaattg caaggctttc agtagtaatt atgggttctg gtataatttt 5940 aaaggatccttcctctggct gtcatagggg ttgggccctg ttgaggtgcc attacaaagc 6000 acccaggaccagtgttagca tgcagggggg aagtatggtt acagtcagga catgttgagg 6060 ggggctgatgggatttgctg gtgggtaatg tagagggaag agttgagggt gactgaagtt 6120 tgaacccccgctctgccagt tgtgctctct gtgtgcccct ggagcagtac tttcccctct 6180 ttggacccagattcctcatc tgcaaaatgg ggataatgat gaagaatcag cgtgtgcctg 6240 gttgtgaagatgcagtgaaa ttggcttgct cattgttctg cggtagtcat atggtgggga 6300 ttgagcagacctgaggaaat ggggcatctg gggaaccgtg tgggaccaag tcagcaagtg 6360 ctaaagcctgggttgggaat acactgggct gtttgttttt ttggttacta tcaaggaagc 6420 agttgtgacaagccaggccc agtgagaatg gatcagtaga ctagggtcag attgtgttgg 6480 gccttgtaggccagagtaag gccttagctt ttattctgaa tgtgcaggga aaacatacta 6540 catgtgggattataccatat acattcatat tatggaaggc tccatgggtt gaatgcaggg 6600 tggatcttcaaaagtgggtc ccctttccac aggtgcatat agtgggaagg cggattttat 6660 ttattaactcaccacagagc aggctctgtg ccgaggcctg ctgggacaca gagagtccca 6720 gtctagggtggtaagtgctg tcatggaacc atatgggtgg actctttgga aaatcccaat 6780 tcctattctctcacttaaca caacaatcaa caacacagaa gaagacttcc gtgactaaat 6840 gtgtgttggggtggggaggg attcttcacc accactaagc aagcattcag ttctgcagca 6900 cacgccaactggatgtcctc caattcagtt cagacacgac ctggagacag tgttggatcc 6960 aacaggttgagggctcaggc ctcaaaactg ccccttggcc ctcgccttca gttgccattc 7020 tgggtcgtcagaacttctga tctacctgct tcaagttgga gttcccatga ccaccctgcc 7080 tttgggtttgattaatatgc tagagcagct ctcagaactc agggaaacat gtaagtttat 7140 cagttcattataaaggatag tataaagaat acagatgaag agaggcatag ggtgagctat 7200 gggggaaggggtgtggagct tccatgccct tcttgggtgc accaccctcc aggaacctct 7260 gtgtgttcggctatgttgaa gttcccaggc cctgtccttt tgggttttta taaaggcatt 7320 gttaggtaggcaggattgat taaacctggt gatcaccttg accttcagcc cctctccgat 7380 ccctggaggttgtggggtgg gactgaaaat cccaaccctc taatcatgcc tctctgtttt 7440 ccatgactatctttccagtg accatggacc ccatcctgaa ggtatcagtc aacattagca 7500 tacaaaaagcgccttggaga ttccaaagat ttgaggagtt gtatgcaagg aaatggggat 7560 gaagcccaaatctgtatctc acagtatcac aactgtagca ggtgcttggt ggggacacac 7620 ttgggagatagtcagtgcac agggaattct caggatatga catgatcaat ggccagtcaa 7680 ggtttctgaaatgatattaa tagattttct atcaggaatg tgacggagac cccaacctag 7740 agttgtttaaacaagtaaga gttctcttta tttctcatat agcaagttca gcagctcaaa 7800 gctatcacagaattgggtca gctcaaagat atcacagaaa tgcggttttt ttgactggcc 7860 tgttttggttataagattcc agcagcatct gctgtcctag tggaactgca ctttttagtg 7920 cagtttagcagaggccagct taggacacag aggttctttc cctcctccct tttatgaagg 7980 aggaaaatctctcctgaatc tcctcagcag acttgcttgt atatctcttt gccaagaact 8040 aggtcatatgcagagttagt tgaagttaag tttggctgta agtagtagaa aaactctaaa 8100 taacaggggcttaaatgagc ttgaagtttc tatctgactg atgaccaaaa tgtctggtgg 8160 caatccaggctgctgtggca cttcatggtg tcagggtccc aggctcttac cattatgcgc 8220 tggttgtatcctccattcta ccctccgtgt cccccaggtt acctcaggtc cccaagttgg 8280 ctgccgaagccccaaccatt atagctccat tccagacagg aaggagggaa gataagtgtg 8340 acccccatccctataagttg cacaaaacat ttccacttgg attttattgg ccagatgtta 8400 gttttatagtcacattcaga tgcaaaggca attcagaaat gtcttttcca ggggtcatgt 8460 gtacagctgaaaaccaagga ttatgaacag cagtatagcc ccatgcagga gctcctaact 8520 tggggatcatctgcaattcc caggcaggtc catgaatttg gatggcaaaa ataacatctt 8580 tattttcattaacctttaac ttacatttaa cttcccttct gttatagatt ataatatcac 8640 tatagtatatcataggtagc gttagcaaaa cctgtgactt accagtggaa atcaggagtt 8700 gtgttcacatgtcatttctg ctattataca tatcttctag tgttgtttat gttcacctct 8760 ttcaaaattgcgttcattat tagacctgct aaggggattc atggcaccac cccacccctc 8820 tacaagagaaaaaccaaaaa aagccgggtg cagtggctca tgcctataat cctagcattt 8880 tgggaggtcgaggtgggagg attgcttgag ctcaggagtt tgagggcagc ttgggcaaca 8940 tagtgaggcctcatccctac aaaaagttaa aaaactagct gggcttggtg acacctgcct 9000 gtagacccgtctacttgggt agctgaggcg ggagaatcac ttgagcccgg gaggtcaagg 9060 ctacagtgagctacgattgt gccactgcac tccagcctgg gtaacagagt gagaccctgc 9120 ctcaaaacaacaacaagcac aacaaaaaag aaaaaacaga gcaaatatga atccatagta 9180 ctggttaagaacctggtggc tgggattcaa atcctggctt tactaccagt aagctgtatg 9240 cagttaagctccctgtgcct ttgtgttctc tgcctcaaga cgggggattc tgtatttccc 9300 tcatgggattgtgagggtca aatgaattaa aacattaaag cctggggcat agtagagact 9360 aaggaaatatttactgttat tattctgtca tggtgaaaaa agggaaaaga aacatcagtg 9420 gaccagctgccacgatgtcc ttcttaataa tctgaacaaa atcaggatgc tgtcagtgag 9480 gcgtggttgccacggctgcc attattaatc ttcccctggt gctagggtgt ccctgtagaa 9540 tgtcattttgtactcaagac aaccctctga gggtaggtat tctcatcatc cccacttcac 9600 agatgagaaaattgaggctc agaaaggtac aaggattgcc caagattcca cagctaatat 9660 gtaagcagtttagccttgaa cctggacagt gcagctctgt agtaccaccc taggccatct 9720 ttctcaagcattgatgatgc tgtctgcctg aagaaacact tttaatgaaa ccaaaagcag 9780 aatcccaaagaacagcagtg gaaggaaggg agctctctgg atgggaaagc agcctgattg 9840 tcatgggcccacctcttggg catccccagc ttcaagttgg ggttcccacg accccctctt 9900 tgggtttgattaatttgctg gagcggctca cagaactcca ggaaacactt aggtttaccg 9960 gtttattataaaggatattc caaaggatac agatgaagag gtgcataggg tgagctatgg 10020 aggaaggggcggggagcttt cgtgccatcc ctggggatgc caccctccaa gaatgtgcat 10080 gttctgctatcaggaagctc tctgaatcct ttcctctttg gtttttatgg aagcttcatg 10140 atacaaacatttttgcctaa atgacagtat gatattgaaa ttgagtcagg ctgcctggct 10200 tcattgctttccagtaagtg acagcctcac ctctgtgcat cagtgtcctc actcgcctct 10260 gtgcatcagtgaagtgggga cgctgctaac aatacatacc tcagagcagt tatgaggatt 10320 cagtgtgttaatcatccatg tcaatcactt ggattgctgc caagcacgta gcaaacaata 10380 aatgtttgttgctattgttc tggcatttat gattatgatt attaattgta attattccct 10440 gtaccatctcattgtcctca gggctgttgc tcacctagta tggccctagc ccatgtgtat 10500 atttccccatagctcaggct gagagaagaa cacacagatc aacttggaga aaacaaagag 10560 gaaagaaataggcaactagg tcaatactcc ccacagtatt ttatgttatt ttattttatt 10620 tatttatttatttgagacag agtctcactc tgtcacccag gctggagtgc agtggcacca 10680 tctcggctcactgcaagctc cacctcccag gttcacgcca ttctcctgcc tcagcctccc 10740 gagtagctgggactataggc gcccgccacc acgcccggct aatttctttt tgtatttatg 10800 gtagagacggggtttcaccg tgttagccag gatggtctcg atctcctaac ctcatgatcc 10860 gcctgcctcggcctcccaag gtgctgggat tacaggcatg agccactgcg cctggcctcc 10920 ccacagtattttattatgaa agttttcaga catgcataac tttggaagac taacacaagg 10980 gtcagcagactacagtccat gggtccaatt cagcccactg tctgcttttg caagtaaagt 11040 tattggaacacagctatgcc tgtttgcttc tctagttctg gttctatggt tgttttcagt 11100 gccacgcaagcagagttcag tagttaggtt tgcaaagctg aaaataatta ctgtctggca 11160 atgtatagaataagttttgc tgacttctag catagtgcag tgagcacctc tatacttaca 11220 ccccaggtcagtgattgtta gtgtttgcta tattagtttt ttatctttct ataggtctgt 11280 ctctatatacgttttatttt ttagctgaaa atcagttgca ggttttgtag tactttatcc 11340 tgaggtccgtttaatgccag aatggtgagt ttttggacta cctgtatgaa agatttactg 11400 tgtgcatttttctgaggaga gaccatcaca acacttcatc agatgctcat cgtctgtgat 11460 cgcagagatattcttcaact cctggaccgg agaacccaac tcacgcaaga tcttagtcac 11520 ttgtagacctcgtctcagtt atgtagggcc tcagaacaga acaggctaag ggagctcctg 11580 ctcctcaacctcgccctgtc cctacctgtt ctgcctctat ccaggaagaa aggtcccccg 11640 tgggaccattagtcactctc cccctcatga aagcatttcc tcccactctc acactcatga 11700 gctcattccattcttgtgac agtgctggga gctattaata ataggagagg cctcgtggtg 11760 atgaagaaatgaggggagag tttcacagtc tgtcctgcca tcagagcagc tggctttgga 11820 tcccacactccctcggtgct tgctttggta ggtgtcttta cctctctgat cgtcactttc 11880 caaagtggtgaaaagaggat attagccatg cttttctttt aggatctgga ggatcaaatg 11940 aggtcccaacatgtaaatgc ttgttgtgag atctattatg tgtgttagcc aaatataata 12000 gttctctaggactgccacag caaatcacca tgaactgggt agcttaacac aacgaattta 12060 ttctctcacagttctggagg ccggaagtct gaaatcaagg tctcggcagg gttggttcct 12120 tttggaggctctgagggaga cccattccat gcctctctgg aagttaggac ttctggtggc 12180 tccagcaattccttgtgttc ctgggcttgt ggatgcatct cctggtctct ccacccatct 12240 tcacatgcgtgccttcccct ctgtatctgt cttcctttct gtctcttaga aggacactgt 12300 cactggattcaaggctaacc ctccgtttag gatgatatta tctggacaca cttaactacg 12360 tctgcaaagactgttttgac gtgcacgtca ctggattcaa ggctaaccct ccgtttaggg 12420 tgatattatctggacacact taactacgtc tgcaaagact gttttgatgt gcacgtcact 12480 ggattcaaggctaaccctcc gtttaggatg atattatctg gacacactta actacgtctg 12540 caaagaccgttttgacgtgc acgtcactgg attcaaggct aaccctccgt ttaggatgat 12600 attatctggacacacttaac tacgcctgca aagactgttt tgacgtgcac gtcactggga 12660 cataactttttgaggctgct attcagcctc ctgtactggt ggtattatta ctactgctaa 12720 tgctactcttgtttttgtag tcattttatt acaataattt ttttttctct ttgagacaaa 12780 gagtctcactctgttgccca ggctggagtg ttgtttgttc ttttacaatc acttagccag 12840 caatcctggctttaaatcct gctccatgtg atcttagctg tctgaccctg agaggggctt 12900 ttgttccccgagcctgtttc cctgtctgta aaatgggact ccagctagtg acatgggtgc 12960 aggtgtgtggatgtgtttag ccgaggaccg ggcacagagg aagcgtgtta taaacgtggc 13020 tgctgctgttaaagtacagt gcattggcat gttacatgat actgtgtata ggagaaagtg 13080 atgaagagagaggaggagag ggagtacagt tttcaacaga gtggtcctgg aaagtgcaac 13140 tgagtaggtgtcaccttcat aagacccaag ggatgctttt gcatttgttg ctgctgccac 13200 tactattgttgttatttaat gattccctcg aggtcacagg gtctcttggt gggtgtttgc 13260 caaattactcttgagcatct tcacatgtaa ccaagcaagg cttccagggg ctgactgggt 13320 tgagagctgtcagaactcac gcgtgtctgg gatttctaac attctcccct cctcttcttg 13380 ttctctcattagcttcgcct caacagcatc aagaagctgt ccaccatcgc cttggccctt 13440 ggggttgaaaggacccgaag tgagcttctg cctttcctta caggtaacaa aggggacccc 13500 tggggcccagatgtggggac tcttgggagg tggttttcac tatataagag aagacttgtg 13560 gatttaacatattgtttgtg aatatctgcc ctgtgttaga cactatggag tgggaaaagg 13620 gattcagagaagtgcagtct tgctctaaaa gaatggtctg gaatgatgga gacagatggt 13680 gaaagggatctagaagcagg tagaacgagg tttgagttgc agctccaaca gttggaagct 13740 ggtgactttagacaagttag tttacctgtt tcttatattt ttcatctgta gattgggata 13800 atcatccatgtgccaaagtg ttgatctgag cattcaatgt taataacaat tgctaatact 13860 tatgatatgcttactgtatg ccaggtggtg ttctacaccg tatcttgtga ggattgagtt 13920 catataaagtgtttagaaag ctgcctgtac tcagtaaaca tacatcacta tcatttcacc 13980 cagttgatctcacagcagtg ctttaagcag gtaatactgc tgtccccatg ttatagatga 14040 agaaaccaaggtgtagagag gtgaagtgat tcatttgccc aaggtcatgc agtgaggagg 14100 taactgagaagggatattta tctagtcatt ctggccttta acttgacagc attggttaat 14160 gactgatttcaaatctgagc tctaccactt tctaggtgtg tgactttggg caagatattt 14220 tacatctgcctgcttcagat tcttcacagg tgaaatgggc ataattatgt aacctaacat 14280 gaagactaaataagatactg taaagtgctt ataacactgt atagtacgtg ccgcctaagc 14340 gatagctgccaggctgttac taagcactgc tgtttgtgag gatgtaggta aagcatttaa 14400 tacactgtctgacacagact ggaccacaat taatagtaaa ttctctgtta tcattattcc 14460 cattactacttcatatttta tacttattac cagctgaaat cacagctgaa ggttcagagt 14520 gctttgtcactaaactcttg tgtatgggaa catcttactt ggacctagtg atgatcctgg 14580 ggaggaaggagcttgagatt gttacccctc cccacactgg acagatgggg tattggagcc 14640 taaagaggtgcagtgactcg cctacagtgg cacagctagt gactgagggg tgcaggtgtg 14700 tctgactggatggtgcatga agagtaggtg tctgctcgta tgacactgaa caaatggccc 14760 caaaccaggtggcttctatt ggtgaactgt tggggagact tgaaagcttt agaatgctga 14820 agcatggcaggataataaag tggtgaagaa gttgtagagg aaaaatgttg gacctgggtt 14880 tgcaataagcagctctgcca ctgacttgct aggaaaacct gagtaggtca ctccacctct 14940 cttagcctcatttcccttgt ctgtaaagga gtctaataag aggagtacct aacactcggg 15000 gtgtgtagggaaaactctgt gactttcctc tactttcaca ccacagcaat cataacacag 15060 aacatgacttctgtgaccat atgtctgggt ttttttcccc aaactaagca gtggacagca 15120 gctggttgtcctctaattca gttctgacac gtctgcccag agacaggttc agatcccaca 15180 gattgatagctcagtcccca ggactgtccc cagccctgac caccagtcgc tggtcctgtg 15240 gaacttctgattgatcagct tcaagttggg attcccacga ccccctcttt gggtttgatt 15300 aggctcatagaactcaggga aacacttatg tttactggtt tattataaag gattttgcaa 15360 aggatacagatgaagagata cagagaatga ggtgtggggg aaagggcctg gagcttccgt 15420 gccctccctgagcccaccac cctcaagaac ctctatgtgt ttcaccatct ggaagctctc 15480 tgaaccctctgggtttttat ggaagcttaa tggcagaggg tcctgagtgg aacggaggca 15540 ccagccatgtggaactctgt gggaagagca tttcaggctg tggggagagg cagatgtaag 15600 ctcagtgtgttggaagaata gcagtgaggt agccatgggt tgagcaagat tgttaaggga 15660 gagtgagactaggacatgag gttggagaag aacagtgacc acagtatgtg agatcaggtt 15720 tctttgaaactattggcaga ttgaaatgaa agagtgtcgg gatttctttt acacctgaaa 15780 gagtcactctggggtctggg agatcagatg gtgtaggggc aaaggtggaa gcaggaaaac 15840 caggtaagaagctgttgcag tctcccaggc gacagctagt gatgatcaga gtgaccttag 15900 tttgggagggttgacaggct tggctcagaa ggagctgcca acccttccaa gaaggatagt 15960 tctctgtggattggtggaaa gacaaggtgc tggcagttcc caggtgtttg gccttcagca 16020 gcattgcttggctataaggt ctagactgga gataaagatg aataagaatt gagtccgtgg 16080 atacgcataaggtcttcagg agacaaagaa aacagggagg gtggttttaa ctgtgtgcag 16140 agggagccagggaaactgag gctgggaacg agcctaagaa cagtgtagct ggagtcacat 16200 tggcgagaatttatttattg aaggaagagg cagagatact aaccctccta ctgggccctg 16260 tgctgagctcttcaatgaaa gcatttattt ccctttagtc tccgaacaac cctatgaagt 16320 gagaacagccctgtggtgat ttaggggatg acaaaggctc agagagatga agtgagctgc 16380 tgtataagcagccaacagta aagcaagatt tgaacctacc ataaaatgga gatgatgata 16440 atgactccttaaatgagagg gttgtggtaa ggaggtgatg cagaagttga acccaccact 16500 tacactcacacccctttggc cagagcaagg ttacgtggcc ttgccaactg caagggcgtc 16560 tgggaaatgcagtcggctgc tggattgcca tacacccaac ttaaatgcag gaggtttaag 16620 tgccaatagatgccagggat attagcagct tgtgctgtgc agagatcagc agttcccact 16680 gcctcagggatgagaggatg gagcttaaga gagaggcctt tggatgtggg cattagggac 16740 agaacggattttcccttcac atattcattc tttcagcaaa cctgaattgc tgtctgtctg 16800 tgctcagctctggcaggaac tggggcacag agaggagcca gtcctgggct cgctgattat 16860 agcctatgttaaggatgtgg taaaggtggc atggaaggag agagagactg agcccagaga 16920 aggtgtagcggacatcctag aaatcttcca agaaggggcg gctaaactga atctccaagg 16980 ataaaaataaatttctggca gagggaacag tgctgagggg agaagctgga ccccaaaaaa 17040 gctctcgttgaccatgctct gcgctttata cacctagtca tatttgctcc tcactgtaac 17100 cgttgctacgagggagggat ggtgaacacc attttacaga cgcagaagct gggactcaga 17160 gaggtggaatcactcagtca gtatttcaca cccgctagag ggcagaagca ggacttaggg 17220 ctctcttatcccagccaagc ctgtacctta atatctgtgg cagcccaggt ggagagtggg 17280 ggagcaagtgggcggatgga agaactgagt gctgacagcc tgaggaagca gaatggagtc 17340 catgtgttctgagcttgggc tggggtcaga gttgagatgg gatagtcacg aagtctgtct 17400 tggttccacagataccatct atgatgaaga tgaggtcctc ctggccctgg cagaacagct 17460 gggaaccttcactaccctgg tgggaggccc agagtacgtg cactgcctgc tggtgagtgg 17520 aaggcaggaagtcctcttgc ccacccctta gggtcggccc atggtcctgc cggcctaggg 17580 cagggaggggagcgtgtcag agagcgtggg gatcacgtca gaacagccgg gccatggaca 17640 tccgctttaatccaacaagt caggagtggc ttttaatatc gtgaactcag ggtgcagtta 17700 tcatcctttctgtttagtgg tcaggtaggt gcccagtgcc aggccttacc tgggattcta 17760 catagggctgtgggtttaga gtcaggttgg gttctactgc ttatttgctg tgtgacttgt 17820 aagagaattacttaccctct ctgtgctgta cctttctcat ctgcacaatg gggttatgaa 17880 aacctttttaaattaccttg aagagcccta gcacagtact tggcacttag taggtactaa 17940 ccgatgttaactgcaattgt cattattgtt gctgtgacat catatccagc ctttggggta 18000 ggagggattccctgtttatg ggtgagccac ctggagcact gagaggttaa gcgtctcttc 18060 agattgtatgactagtagat ggcagagctg ggattcttgg tctgtccatt tgactgtctg 18120 ccaggtccccaccccactgc ctgcctgtgt gtcccgtgct aggtgatggc caaggacaca 18180 aagatgggccagatcccaac aagcagcgtt gtttgagaga agatgatctg ggaaggcaga 18240 ccgtacaggttacctagtcc cctggttttc actgttctca gagtgctggg ccactgctgc 18300 tgcctctgcctctgccccac tcccccagtg attgtgtatt tcactgtcgc tagagataca 18360 atgtggcaccggcagttgat ggtgtattag ttagcagcag cctttctcct agctctagtg 18420 gtgtataaagaatggtgctt atcattgttg ggatcttggg atttgagtaa atataacaat 18480 ttgtttcttcccaaatagca ttatgtaaag aaacttgata gttaccatta aattcggata 18540 tctcagccttcatctgggca tatgtgtata atttgtgcgt atggacacac agaaaattgt 18600 atacaaatagatcgtttcat acattttaca caaaacaggg cttaacatga gcatgatgag 18660 gtttgggggccagcctagaa ggggttcctt gaatttcaca aaattatctg tagcccctgc 18720 ctggtatttgtcaggtggaa cttctgagca gtgaattcat agctttcagt agcttctcag 18780 aggacttaggagcagaagac attattttta tgaaaccgca aggcttcaac aagggccccc 18840 agggtctgcaactgaggttg ggactcaggc ttgctgtgga gctgggctgg gacccaggga 18900 tccttctcctacctggccag ggtctcccaa cagaacattc tcagtgagga aagtcccagt 18960 caagccacgcttccttttgg gaagcagttc cctgtgaccg atgtgcctga cgttcatgtc 19020 agccagcagacacatcctgg ggctgtctgg gccaggcagt gctggaaacc agagaagagt 19080 tagatcagaatagcttgtgc ttgctcgttg cctgcagtgc acatcccaag agccttacac 19140 agttgtatactgatgttagc tgtattgccc agatgaggaa actgaggcac aggcagattt 19200 aagtgaattgtccgagctgg gaagaggcag agtccagatt cgatcccagg cccctaattc 19260 ttagccactcctatgtgcta tcctcagagg gctcctggtt gctttcagag ctgtaaacaa 19320 gggaggcaggggggtcctta aacccaggga aggcgcctaa cctgcttagg tgggatggga 19380 agttgtcagggaaggcttcc tggaggaggt ggctgttaac ctggattttg acaggttata 19440 agaaatagaaaatggctttg tgtagtttct tcattccaca aacattgagc aaggtctcta 19500 ggctatggaaacccagagat gtgtcagacc cactcccggc ccttgctctc agcagtggtg 19560 aaatggaattaattacagag actcctgtag ctgcagtgta aggattgttt ttttgtttgt 19620 tttgtttttttcccgagatg gagtttcact cttgctgcac aggctggagt gcagtggcgg 19680 gatctcggctcactgcaacc tctgcctccc aggttcaggt gattctcctg cctcagcctc 19740 acgagtagctgagattacag gcgtgtgcca ccgcacccag ctaatttttg catttttagt 19800 agagacggggtttcactttg ttggtcagat tagtctcgga atcctgacct caggtgatcc 19860 acccaccccggcctcccaaa gtgctaggat tacaggtgtg agccaccgtg cctgggtgtg 19920 agggttgtttctgtgtcagc agctcttttt atttttattt ttctttgaga cagagtctcg 19980 ctctgtcacccaggctggag tgcagtgttg taatctcagc tcactgcagc ctctgcctcc 20040 cgggttcaagtgattctcaa gcctcagcct cccaagtagc tgggattaca ggcacccatc 20100 accatgcctggctaattttt gtattttttg tagagacagg gtttcaccat gttggccagg 20160 ctggtctagaactcctggcc tcaaatgatc acctgccttg gcctcccaaa gttctgggat 20220 tacaggcgtgagccaccgca tccggccagc agttcctaac tcttttggaa ttatggatcc 20280 ctttgagaatctgttaaatt ctggaccctc ttcttataaa agtacacagg attttaggcg 20340 gttcacactcttccctaccc tgtgtggtct ccaggtttga gaacccttgc ccccagactt 20400 cacagagaggcttcacagta gtgagtcttc actgtgcaaa ggcatcaatt gcagtttctt 20460 aaatgtgtaccttcctgggc cctgcctcca gatattctga ttgttttggt ccatgctggg 20520 ataggcctccgggtgattct gctgcagggt ggtcagggat catcctttga gaactacttc 20580 ctgcagtaacctacaggaga agagaacctt ggcttacata attactgctc agcctctgtc 20640 ccaggcctccttataacaga ttgccgtaag ttccaccggt agtcacttgg caagtgttga 20700 ttgagtaccttctctgtgcc agcccaggtg gtgggagaac tgaggtgaac aagaggaact 20760 ctgtccctgcagtatcccag tcacaccgca cctatggtca cactcccatg atactgtcct 20820 gaacttgagacgtcacgata gcatgaggac ctgtgacagt gacattatgc tggcttgctt 20880 agtattacatttttcctttg aatcattcat tgcaactatt tttaaatgct atttttaaac 20940 atttgtttttattcttagtt ttactgtaaa aatatacata gaatgtgcta ttttaattat 21000 tttaaagtgtgtagctctgt ggcggtaggt ttattcacat tgtgcagcca ttatcaccct 21060 ccatctccagaatttttcac ctcctcaaac tgaaattcca aacccattgg acatgagctc 21120 ccccttccctctcccccagc ccctggcagc caccgtagga gtttgactgt tctagattcc 21180 tcatataagtggaatcagaa aatatttgtc tttatgacta ttagctaatt tcccttggca 21240 taacatcttcaaggttcatc catgttgtag catgtgttac atgagacttc tcacatacaa 21300 gattcttgagtttttttgag ggctgcattt ggaaaaccag atttgcttat tgccagctgc 21360 attctcccatagtgacagtc cttggagctg gggagcacct gccctccctg ggctacatgc 21420 cctccagggtgccacagtgc ctgcctcttc ctattcatgc ctcctgccac ccctgcctgt 21480 caccttggctgatggcatca ttctgttact tgactgagcc ctggaggcat ttccatttat 21540 gattttttttttcctgcttt aaatacctgt cagcccaagt tgaatttcag atcagagacc 21600 tccacacctttatttgaaca tattgggata agttgagtct cccttcagta ggtcactaat 21660 taagagttcctgtcttgtgc taagagtgct gctgggctct ggggatacag tggagagcca 21720 gtcgtatgtatctgaaggag cacagtcatt ctaggcccac agtaacaacc agcgagcagt 21780 cccgagccccatagtccccc agaaacatga gatcccaatt cagtcaccag agctgtgtgt 21840 aactgttcatgggaagctta ggtcaggttt tcgatcctga cctgtagcta ttactagctt 21900 gggcaagccaggctgtacct cagttttctc ttcttttttt tttttttttt tttctttttg 21960 agacggagtctcgctgtgtt gcccaggctg gagtgcagtg gtgcgatctc ggcttactgc 22020 agcctctgcctcccgggttc aaggattctt ctgcctcagc ctcccgagta gctgggatta 22080 caggtgcgcaccaccacacc cagctaattt ttttgtattt ttagtagaaa cggggtttca 22140 ccatattggccaggctggtc ttgaactcct gacctcgtga tccacccgct tcagcctccc 22200 atagtgctgggattacaggc gtgagccact gcgcccggcc cgttttctct tctttaaaat 22260 caggacttgctttatagaat tcagtgagga agaacaagca cccctggtac aacacggagg 22320 tcctgcctagtacacacagt gagtagatgt tctacacagt gtcagtgatg atcattgcac 22380 tttgagaggctaagaacttt ctcctcagaa agcaacgtgt gtaattttaa ggtttatgga 22440 ccccagtgaaactccttcaa ggacccctaa ataagaacct ttgtggaagg cacgctgaca 22500 gagaccttctgctggctggc ataatattac gtttttcctt tgagtcattc attgcaacca 22560 tttttaaaggctattttaat gaaggtcggg atgggtaata gggaagtttt ctctgaggag 22620 atgagcccatgatggggtgc aggatggggc tccagggctg cggatggtgg agagggagct 22680 gtccagtgactttgtgttct caccacagcc accgctggag tcgctggcca cagtggagga 22740 gacagtggtgcgggacaagg cagtggagtc cttacgggcc atctcacacg agcactcgcc 22800 ctctgacctggaggcgcact ttgtgccgct agtgaagcgg ctggcgggcg gcgactggtt 22860 cacctcccgcacctcggcct gcggcctctt ctccgtctgc tacccccgag tgtccagtgc 22920 tgtgaaggcggaacttcgac agtgagtctc tgcctccttg gaagctccaa gctcccatct 22980 cagctccaaccttctctaaa gcctcagact ccttttggtc tagctggggc ccaaatgccc 23040 ctgaactctctccactccca ctcctgctta ccacctgata ggccacatcc tcgagagttg 23100 gtctctggacacggccacgt gtcagtttac ccacctctgc ccccttgctc acttaggaat 23160 tgagatgatgacaggtcctc cttcccactg gttaatgtga ggatttaaaa gaattatcac 23220 acataaagtgcttagagcaa aatctggaac ataaaaactt tcagcaactt acatctgatg 23280 gtatctccagcctgtcccag gtccagtgcc tttggcagat aaaccacctc agttttcagc 23340 ctcctgctcgtctactttgc aaacgattga ccgtcaagcc cgggtttgag cctgactcac 23400 tccagaactctggtttatgg ctgtacactt gcttagatac ttacactggc ccccaccgcc 23460 tttgatcgaagcaattgctg ctgaaaaata aagcctttct gtggctctag acctgcctct 23520 tagttaagcagttttttgtt tgtttgtttt ttgagatagg atctcgctct gtcatccagg 23580 ctgaagtgcactggtgcagt catagcccac tgcagcctca acctcctggg ctcaagcatt 23640 cctcctgccttagcctctca aatagctggg accacaggtg tgcaacacca cgctgggctc 23700 attttttatttttggcagag atggggtctc actatgttgc tcaggctggt cttgaactcc 23760 tgggctcaagcaatcctcct gccttggtct tccaaagtgt tcagattaca ggtatgagcc 23820 actgtgcctggctcgttggt taagcaattt ttatgggaat gatgtaatcg tcagcactta 23880 gcattgactagatttattat gcgcaatctg cgaagtgtct cacacacact attttcattt 23940 aaacctcatgcggacctgtg gggtaggtac tgttactatc agctccgttt catagggctg 24000 ggaagacagagagggggtca tcacttgccc aaggtcattc agctaaaacc tggacccaca 24060 caactgcagagtctgtgctt gctcctctct gccatactgc ctgctgcctc aggatccccg 24120 tccccgactcccaggtactt ccggaacctg tgctcagatg acacccccat ggtgcggcgg 24180 gccgcagcctccaagctggg ggagtttgcc aaggtgctgg agctggacaa cgtcaagagt 24240 gagatcatccccatgttctc caacctggcc tctgacgagc aggtgagttt tgcttcctgg 24300 ccctctgctctcccgtcctt ctggtggttc ctgcccatga aagagaatcc cagagctcag 24360 caaggcctctgctgccctcc cactgttcct ctcctctccc taggactcgg tgcggctgct 24420 ggcggtggaggcgtgcgtga acatcgccca gcttctgccc caggaggatc tggaggccct 24480 ggtgatgcccactctgcgcc aggccgctga agacaagtcc tggcgcgtcc gctacatggt 24540 ggctgacaagttcacagagg tagatgagcg accgttgaca ttgtcccact ggtggggaca 24600 ctgacactctcagaagggaa gcatatagga gctgaggttt ccattaggcc gatggaacca 24660 ttgggcgtttgagcaataag atctctatga tcatctaact gcgtctcgct tcgtgtgcca 24720 atcctggttgattgacatgg catcttaaag tgctgccttg agaaagattc tgaggcaaag 24780 ttaaggctacgtggaggaaa gtgccacagg agcagagaag ggtagcacat gtggggtgtt 24840 cctgacataatcaagctgtc ctttcacaaa ggggaagaca gcccaaaaag gtggggtttt 24900 ttggtgtttttttttttttt tttttttttt ttttttaaga tggagtctgt cgcccaggct 24960 agagtcttgttacccagctg gagtgtggtg gcgcaatctt ggctcactgc agcctgtccc 25020 tcccgggtgcaggcatttct cctgcctcag cctcctgagg gactgggatt acagatgccc 25080 accacgacacccggctagtt tttgtatttt tattagagac ggggtttcac catgttgtta 25140 gccaggctagtctcgaactc ctgacctcaa gcgatccgcc tgccttcatc tcccaaagtg 25200 ctgggattacaggtgttagc caccgcgccc agccccggaa agtttaatta actgatcaga 25260 gtgacactaccagccaggca gaaaggggac aagactccag gtctgtgact ctcaggacag 25320 tgctccttccacagggatcc agattgcctc atcccacaaa catgtttgct gagcaccagc 25380 tatttgctgggccagtgaat tcggatcatt cctggccttc atggagctag gcagtctgaa 25440 ggggaagactgacttagggg aaatttgatt ataaagtgtc acaggtgtgg gacagacaga 25500 cagatgtggggccttggaag cattgaggag gggaggtggt gttacagctg gttctagaag 25560 atgagtgggtaagagctaag ataggaactt tgttccagcc agaggacaaa gaaccctggg 25620 aggtgagagcaagtgcaagc aggaacattc aggcctgatc ttgatggccc agcctgagag 25680 aaagcaggagagagggcagg gtgggatcgg agagagggca gggtgggatc agagaggcct 25740 tgagtgccactccaccctga ggcgcccttt gcctttaatt atgctggttc ccactggcat 25800 ttgcgggaaggactcagagc ttcagaatag cgtaccatca ccacagttag ggaaggttct 25860 tcccatccttgtctcctgag ctgcataaac tgtgtcacac tgggtcttag aataaaaatt 25920 ccatgagggcaggaatttta ggctgttaaa ccagttcttg gcacatagta gacattcagt 25980 aaatatttgcaagatgaata aaaggcagta ttttcccaag atatcatgag gtccttcaag 26040 atttttacttgttcattccc gtcttcataa tgaactgtcc tgcttcctac caggtcttca 26100 ggaaccagctttgcagcagg agccgtgcgt ctttccatgc ctggtgccat gaaacaggca 26160 gggccaagcgtgcctccctt tttttttttt tttaagacag agtctcagtc ttttgcccag 26220 gctggagtacggtggcacaa gctcagctga ctgcaacctc cacctcccag actcaagtga 26280 ttctcgtgccttagcctcct gagtagctgg aattacaggt gtgcaccacc acacccagct 26340 aattttgtatttttagtaga gatggggttt caccatgttg gccaggctgg tctcgaattc 26400 ctggcctcaagtgatccacc cacctctgtc tcccaaagcg ctgggattac aggtgtaagc 26460 cactacgcccagccctagag tgccttcctt tctgtcaatc tttattgttt tatttttatt 26520 ttgagacagggtctcacacc atcacccagg ctggagtgca gtggcacagt cacggctcac 26580 tgcagccttgacctcctggg ctcaggtgat cctcccactt cagccttctg agtagctagg 26640 acgataggtgcctgccccca cacccggcta attgttttgt tttttttgta gagtcagggt 26700 ttcactgtgttgcccgggct ggtcttgttc tctgggactc aagcgatctg tccacctcag 26760 cttcccaaagtgctgggatt ataggcatga gccatcgcac ttggcctatt gttttatttt 26820 cattacaaaagtaattcgtg cttctggtaa cagatcttta agaaatacag ccatatataa 26880 atcaaaaagttgcagtcact atatcatttg aatgtttttg aatcaggtaa cagaaaactt 26940 aacctcagtagcctgaataa tatggaaatg tgttattttt aatgctgaca acaccgtgag 27000 gtaggtgccctcgcagtcct catttttctt tggaagcaaa agaggctcag cgaggtgaag 27060 agccttgccccggggccagt cttggcactc gaattagatt ttagcactgc ttccaaggcc 27120 cacgctctgtcccctaattc tggtgccttc actttgattt tggcttcctt agcccagagt 27180 aaactgccagcccctctcac tctccccctc ctccttcctg tctgcagctc cagaaagcag 27240 tggggcctgagatcaccaag acagacctgg tccctgcctt ccagaacctg atgaaagact 27300 gtgaggccgaggtgagggcc gcagcctccc acaaggtcaa aggttggtgc tggcagccgg 27360 aacacagcaagtggggtggg tatccaaggg gctggaggtg gaactagcac atcaggtctc 27420 acttccctttgcctccctct ccctgcccac agagttctgt gaaaacctct cagctgactg 27480 tcgggagaatgtgatcatgt cccagatctt gccctgcatc aaggtaacag agagtttgat 27540 gggaggaaccaagtggatcc gagcctgcca aaaagagggg ctggagacaa ggctttgggg 27600 atagtcagctgcaaactagg ttcccagccc tctgggacca ggcagctctt gggtttcaag 27660 cagttaggggtcctgactgc agcttgaggc tgaccttaaa ggtggaagta ctttctagaa 27720 cctcagatgtcactgagtcc tgtcattcac agggttttgg ggttggagtg ggggctgctg 27780 agagcaggggtcattgaact cttaagtagg tggtactcat aaggaatagt gatttcccct 27840 gtaccctaagccatcccctg ctctatgaat gagaggggca gaagcaggtt attgtctctt 27900 aggagttggcatctgcttag ccacttgctg ctgcaggggt tgcactgacc cctgtgcctg 27960 cctcttctctctcccaggag ctggtgtccg atgccaacca acatgtcaag tctgccctgg 28020 cctcagtcatcatgggtctc tctcccatct tgggcaaaga caacaccatc gagcacctct 28080 tgcccctcttcctggctcag ctgaaggatg aggtaagggc accaggatct cagctctggg 28140 tttgtggaggggacaggcgg gtcttcctag attgctaggg tttacctaga ttgaccagga 28200 atctgctgatatctcaacag acatccagat ctttgctgag ttgcatgttt gtgggcatag 28260 ctgtgtgttcatgcgttcat tcctccaggc actcttcatg aggcctttcc tggacatgga 28320 ggatatgaaaaatagaaatt taaagttttt atttatggcc aggcgcggtg gctcacgcct 28380 gtaatcccagcactttggga ggctgaggcg ggtggatcac ctgaggtcag gagttcgaga 28440 ccagcctggccaacatgatg aaacctcgtg tctgctaaaa atgcaaaaat tagccaggca 28500 tggtggcgagtgcctgtaat ctcagctact cgggcagcta aggcaggagt atcacttgaa 28560 ctcaggaggcagaggttgca atgagccaag attgcaccac tgcactccag cctggacaac 28620 agagcaagactctgtctcaa aaaaaaaatt atttatttat gttttgagat agggtcttgc 28680 tctattgcccacactgcagt gcagtgatgt gatcatggtc tactgcagcc tccaccttcc 28740 aggctcaagtgatcctccca ccttagcctc ccaagtagct gggactacag gcaagagcca 28800 ccacatctagataattttaa aaaacatttt ccatagagac aagatattat gttgcccagg 28860 ttggtcttgaactcctgatc tcaagcagtc cttttgcctt ggcctccaaa ggcctgggat 28920 tataggcgtgagccgctgcc cccagcctag aataagagtt ttgatcccca aaagccttca 28980 gagactggcagtggagagag acaggcagtc ccatgatgcc attaaggtgt tacaggtgct 29040 gttaaggatgagttcatgtt ttttagggtt taggctaagg tgctctaata tagaccccag 29100 aatacattctggcttaaatt cggtggtgga ttttttttct ctctctcata acagtctagg 29160 acgagattcctcacatgtgc ttgcacccca tacacccatt ggccaggagg gtcacgtgac 29220 cacacccagcagttcaggtt gcctgtatta caaaggatga gaggcggtgc tgggtgatga 29280 ctggagagatcatcagggtg accactgggg aaggagtttg gacttgactg tgggcaagcg 29340 gaagagccagaatagagttg gtgttagaag gcaccctgag gcttatgtga aggaaggatt 29400 gaagtgaggtggccagcagc agtgtaggca gaccaaaccg gaggctgtgg gagtctggaa 29460 ggctgaggctagactaagtg gtatgtagag atgaggctgc attgatacgg aaggattcaa 29520 gattgttcaggaggcagaag ggaccacgtg gtggtttttg ccttggtggt gaaaaactgg 29580 gcagatggtgcaattgtgtg ctggtgtgtg acatctttcc caaaagatgg gaagtgtgtg 29640 aggcttcccaaaagcctcag tctttcttcc ccatcccctt ctttcttttt tatacacagg 29700 cgcccacacagtctgctaga aagttggagg aaatactgta acagaaagta ctgcatcaca 29760 tttcagtcctccatgcccct aaaagttacg ttattcaatt ctgttacagt ggagtaatct 29820 cttagccccagaattacagt gaatttttta atacattgaa aagagtgcgc tatttttaat 29880 atttttttgttctttttgtt tttccctatt agtgggtgat taactgtact ggttttaatc 29940 agttaattacgtcagttgct aaaagtctga atcttcatta gtgttcatga caaattttat 30000 gacttaagtaatttatctga gttatgcttc acatgcacat agtttttata attttataga 30060 tattaatacaatttatattt gtttttgatg ctttaatggg cataatttgg caggtgagag 30120 cccctcaagttgtctgctat gtcatttcac acctctccag cgttttgggg atcattttgc 30180 ctctggcatgagggaaggat ccaggctcag cccagtttga aggcaagccc atacgtgctg 30240 caagtgcgctaggactggag cattttttgc tccagtttca ccttcagcaa gcgctatatg 30300 gtaatcgtgtgctggcagcc agcctgtctc agggcagctt ctcttagaag aaaagaacca 30360 gcatggttttcttggtgtga ggaattcatc tgacttatat ttgagatttc cattttagat 30420 ttataaactttatattttac attttgactc tcaccagaat ttataaactt gtctaaatga 30480 aaaaggctacctttttactg gtgcaccaaa aataagtctt tttaaatggg tgaggtatag 30540 tctctgagccttctcttctc atgcacatgt tcagcagctt gaggctcaca cagcaagggc 30600 tggagctgggcaagggccag tcctatcctc ggaatgtgca gggtttcaac agcccaagcc 30660 tgccaggcttgttcacttgg ttattgattc atttcagtgc tcctttattt atttattttt 30720 ttagactaaaatagttttaa taaggaaata gttttcttcc ttcccttctc ccatgttgtg 30780 gattgatacccagaaaggaa cccttgttag caagcagcgg agctaggatt agaagaatca 30840 tgtctgcctgttttttccag ggtgctgtat tttcctactg tgattcttaa agtcttttca 30900 aaatggataaacatttcttg tgatactatt gtaaggttta aaaatctgct taagttagtg 30960 atccatgctgcttgcttttt gtgtgccgtt aatgtgttcc cagaacgggg agctgggctt 31020 ggacaggagtagtccctcgg gagatgtcca taaaagttga tgcagctgag ctctttccat 31080 cctgtcctgggttgctgtgt gcattgcatt ctctcagaat ccttctttcc tctcctcagt 31140 gccctgaggtacggctgaac atcatctcta acctggactg tgtgaacgag gtgattggca 31200 tccggcagctgtcccagtcc ctgctccctg ccattgtgga gctggctgag gacgccaagt 31260 ggcgggtgcggctggccatc attgagtaca tgcccctcct ggctggacag ctggtgagtg 31320 aggaggcctgggggccaggc agtgctgcct caggggaggt gcagtatgtc cagggctgtg 31380 atggggaaacggggctttga aggcttagtg gaggctgtga caactgcctg gggagtcgaa 31440 ggaagggacccaggaaatag ggccttaaaa gatgcattgg attcaataag agagaggagg 31500 gaaaggagcacctcagacaa agttgagaag tgtccggtct ttctagggtg ggtgtaggtt 31560 ccatgggatgtggctagcag ctccccctgt ttgctctcct ggaacgctta ccttggaacc 31620 cttggtttctcctgtaggga gtggagttct ttgatgagaa acttaactcc ttgtgcatgg 31680 cctggcttgtggatcatggt gagtaccttc acaggagcag caagaggaga tgggagctcc 31740 agaaaggcaggatggattgg ctggggctgt ggcgggcagt ggaggaggct agagtcactc 31800 cccacgccgctggatgctcg tatggaccag ctcgcatgct tgttagagtc ctagagaagt 31860 gttgagtgggaggaggacga aacagatcac ccaggggttg cctggtatag tggagagcca 31920 gagggccactgagcagccag accagggttt tgaatcctgc ccttggggct gtcagatcta 31980 aaacaagtcacctgctgtct ttgaatgagc cccacactca ttctttgaaa cttcttattc 32040 tggtgccttggggccattat gagaatggct cattgtttac actaagagag gtaaaaaata 32100 aaaggtaatatttatctctg tgaagccctg tttgaagtaa taagtgccac ataatttagg 32160 caaatcacttcttagagcct tagtttgccc atctgtaaaa tgaagccagt agtggaacct 32220 acctcttggagtggttgaaa agatactgta taaaaaaaaa cagttactag atatagcaca 32280 tagtaagtccttttttctct ctttggctca catgcagcct ggcacctagg ggctgctttg 32340 taagccatggtgagtgtgac ctacattttg cccacatcag ttcttcacct ccaaatccct 32400 gtctctctcaccctcaccct tctgcagtat atgccatccg cgaggcagcc accagcaacc 32460 tgaagaagctagtggaaaag tttgggaagg agtgggccca tgccacaatc atccccaagg 32520 tcttggccatgtccggagac cccaactacc tgcaccgcat gactacgctc ttctgcatca 32580 atgtgagccttccacctgcc tgctggccca tccctaggga actggagcgc gtgggagagg 32640 agggatgctagagggttccc caagggagac acctggcttg ggaatggaga catggagtgc 32700 atcttctatccagagatgag tccttgggga actgcaggca agggggtggg gctcccaggg 32760 tcagggtcatagggcctctg ggactgggga cttggatggt gagggaccca gggcctggga 32820 gacttgacctgttggagcag cgatctcaag ctttatgagc acccctctcc ttccctgaga 32880 aatgtgtgtctctatctgtg gtgcgttggg tgagtgtatg ggctctaggt cagacctagt 32940 tttaaattctggtactgcca tatattagct atggaccttg gatagttacc taaccgatgc 33000 tttggtcttctcatttgtaa ataattcata ctgtaaagaa ttcatactgg taatcacagc 33060 ctggtgagcgtgaagattaa tcaggttata cacgcacagg gcttagaaca gttatgctac 33120 aggtaggaagtgctcctgtc tatttgcttt tcctacgatt ataattatct catgtactac 33180 ttatttatgtgtaaaccata cacagggcta gaaaggaagg gatttaaaaa taaatataac 33240 taagtgttctgatcatttct tcccacgcca ctctctggag agcattgctt taaggaggga 33300 tcctagggcttggtaattaa ggtcgatctc cagaataatt aagggaagcc tgaggacaga 33360 gaaactgggacatggtgtta ggatggtgtt agtggagttg ggagattcac tccactaact 33420 gagtcacccgtattgctcag cctctgtggg gcctgatgat caccagagtg gcctggtcag 33480 aggcagcaggaaatgagagt tagccaggag ctttgcatac tcacccctgc cactcactgg 33540 cccccaggtgctgtctgagg tctgtgggca ggacatcacc accaagcaca tgctacccac 33600 ggttctgcgcatggctgggg acccggttgc caatgtccgc ttcaatgtgg ccaagtctct 33660 gcagaagatagggcccatcc tggacaacag gtgaggtctg gatactcccc cacacactgg 33720 caggggcttcttgtgggcac cttaatcttt gacctttgaa ggtagagccc agggtcagag 33780 gcctggcagcgctccttgct tgctgtgtga ccttggctcc cttcccttct caaggtgtgt 33840 tttctcaactgtaaaatgaa catcacagca tgaaatagaa agagggggtg atgggttggc 33900 agtcctgtatactagcaaca agtcattaga aaatgaaatc accttatgtt ttatatgtaa 33960 gtatgtgtataatttataat tgcaccaaaa atatcaaata gccaggaata aattcaatga 34020 aagatgtgtatgtaacacct ctaatgtaaa ctgtaaaaca ctaatgtgag aaattaaagc 34080 agacctgaccaaggtgggcg gatcagttga ggtcaggagt tcacgaccag cctggccaac 34140 atggcgaaaccccgtctcta gtaaaaatac aaaaaattag ccggtgcggt agcactcacc 34200 tgtaatcctagctatttggg aggctgaagg caagagaatt gcttgaatcc aggaggtgga 34260 ggttgcagtgagctgagatc atgccactgc actccagcct gggcgataga gcgagactgt 34320 ctcaaaaaaaattaaagcag acctacataa tattcacgga tgggacagtt ccctgaactc 34380 atctttagattcagtataag cccaataatc ttaacaggtt ctttagtgga aaattgacac 34440 ttctaatgaacaaagttgtc tgatttacac taccagagac gaagacttat gacaccacaa 34500 taattaaaatagatgtgaac acaagcatat ttaaatcgcc caatagaatg gaatcaaggg 34560 tacagaaacagtcccacatg tgtttaacag taggcatctc tgcagttcag tggagaaatg 34620 tatgttttttaaaaaacata gctgagtcaa tttgatatcc atttaggaat aaaagaaggc 34680 tcgcctcaatgcataaacaa attaatttga gatgacagac cagaaccaga aagattttaa 34740 aaataaaggacctagaagaa aatgtaggaa aatatcttct tgagttagcg taggcacaga 34800 tttgttaaacagcaaaccag aggaagtgat gggtaagttg accttctgta aaatgtaacg 34860 ctgtttctcaaaagacagca ttttgagagt aaaatgcaag ccactgactg gaggaagatg 34920 ttttaatatatgtttctgag aaaggactca tccacaatac ctgtctacaa atcaggcaag 34980 acaagaaagagatggggaaa aagacttgaa taggcacttg aaaaaggatc tccaaatagc 35040 cagtaagcatatgacaaggg tgttcagcat cattagcctt caggaaaatg caaattaaat 35100 ctcagtgacatatgactaca cgcctcccag aacagccaac attaaaaaag actcagtggt 35160 gcaaatggtgaagacataga agagctggaa ctctcatcca ttgcacgtgg ggctgtacat 35220 ttagggtttgatcactctga aaacgggagt ggatctggag tggaatttgg tgaagcttgt 35280 tatacaaacagcctgtgaaa cagcccttcc actcctgggt ctctatccag gagaaatgag 35340 tgctgtttctgtcagaagaa tgttcatggt agctttattc atagtagccg taaaaatgga 35400 aacaacctccatgtctgtcc acagtagagt agataaattt tagttcattc aaacagtgga 35460 aaactattcagcagtgacga aacaaatgcc tgctataagc agcaacaggt gactctcaca 35520 gataccatgttgagtgagga gccaaaatca agaaaacaca ccatttatgt caagttcaga 35580 aacaggcagaatgaaggaat cgagatgatg gaagtaagaa tagtggttat ttggggagaa 35640 cagggaactgtcaactgaaa gtgtgggacc cacaactgca gatttctctt tctgtctgtt 35700 ccaggacacaacctcagctt tagtttctct ccgaagtccc cctccgtttt ccaaaacaat 35760 tgactcttggtgcaggcgga tttccctggg ccccatagat gagagtggat gcctcctctg 35820 ggctcctggaggaccaggaa cttccccttg gggccactta ccactcccct cttacgtacc 35880 agtttggtctcttcctcctg gcccgggtgc ctcatggcag aaatcaagag tgatttagct 35940 ctgtattcactcctaataca ttgtaggcct cagtgaatat gtgtgaaatc aatgaaagat 36000 acccatttgctgggcctcag agacttgggg gttctgagat tctgttccac tttcccagcc 36060 tgctctgatctccctgtttg gggccccagt ccttgtttat catacttctg acctttaagt 36120 aattggttggttggttggtt ttttgcagtt tgtttggttt tcctttgagg cagaatctcg 36180 ctctgtcgcccagactggag tacagtggtg cgatctcggc tcactgcaac ctctgcctcc 36240 tgggttcaggcagttcttgt gcctcagcct ttcaagtagc tgggattaca agtgtgcacc 36300 accatgcttggctaattttt gtactttgag tagagatggg ggttttccca tgttgtccag 36360 gctggtcttgaactcctggc ctcaagtggt ctgcccacct tggcgtccca aagtgctagg 36420 attacaggcttgagcccccg tgctcggcct tgagtttacc tttttttttt tttttttttt 36480 tttgagacggagtttcgctc ttgttgccca ggctggagtg caatgatgca atcttggctc 36540 accacaacctccgcctccca ggttccagca attctcctgc ctcagcctcc caagtagctg 36600 ggatcacaggcgtgcaccac cacgccctgc taattttgtg tttttagtag ggacagggtt 36660 tctccatgttggtttggcca ggctggtctc aaactcccaa cctcagatga tccacccacc 36720 ttggcctcccaaaatgctgg gattacagat gtgagccacc acgtccggcc tgattttact 36780 tttaattgactcataatttt atatatttat gggggtatag tagtgtttca ataatgtgtg 36840 caaatgtgtaatgatcaaat taggataatt agcatatcta tcaccttaaa tgtttactgt 36900 ttctttgtgatgagaacatt caaaatcttc tcttatagct gttttgaaat atgcaaaatg 36960 ttattattaactatggtcac ccgcctgtgc agtgatcaga gatttctaat tcctgtgtgt 37020 gccctggattcttgagactc ctcccacctt gggtttggtg tatccgtgtc tgtgtacact 37080 ctcttgcccaagagccctgt gcccacctgt tgccccagtc catcctggct caccctctct 37140 ctccctgtctcctttcgctt tccagcacct tgcagagtga agtcaagccc atcctagaga 37200 agctgacccaggaccaggat gtggacgtca aatactttgc ccaggaggct ctgactggta 37260 agacctagaaagcacggagc cctagcagga gggtggactt tgaggacagg cactgggcct 37320 gtgggcagcagcttctggga gggggaggta ccttggcatt gtgggcagag agagggctct 37380 ggttctgattcttgcctgtt cctgttttcc tagttctgtc tctcgcctga tgctggaaga 37440 ggagcaaacactggcctctg gtgtccaccc tccaaccccc acaagtccct ctttggggag 37500 acactggggggcctttggct gtcactccct gtgcatggtc tgaccccagg ccccttcccc 37560 cagcacggttcctcctctcc ccagcctggg aagatgtctc actgtccacc tcccaacggg 37620 ctaggggagcacggggttgg acaggacagt gaccttggga ggaaggggct actccgccca 37680 cgtcagggagagatgtgagc atcccgggtc actggatcct gctgctgtaa tgggaacccc 37740 tcccccatttacttctccac ctcccgtcct ccccatcatt ggtttttttt tgtgtgtcaa 37800 ctgtgccgtttttattttat tccttttatt ttcccccttt tcacagagaa ataaaggtct 37860 agaagtagttggtcctctgg ccttagtcat cagcttggag gagggggcac caccccagag 37920 ccacaacatctgcgcttttc tctggagaag atcttgttac aggacccatt atacccctat 37980 gtcccgaaagaatactctgg ggatcccccc aggagtgctg ctggcctttg gggtagaggg 38040 tccatgaggtgctctgggtg gtgtcctgta gtgcagttca gattcataga tgtcggccga 38100 gtatttggggggttaagagc aggtgccttg gaaccagact gcctgggtcc aaattgtggc 38160 tccttcagttaatagacgtg tgactaggag tagcttgtta agtctttatg agctcagttt 38220 tgtattgtgtaaaatgagag ttacagtaat acctagattg cagggtgggt ggcgaggacc 38280 aagtgaattagtacctggaa agtgtgtaac agtttcaaca tgagaagtac acaacatgag 38340 aagcagagttagctgtacat tgccagagaa ccccctgtgg gccatttcct gtgttcttga 38400 agaagacttgggcttggacc ctgtccccag gaggtctcag agtaatcagg acggtactag 38460 ggagagaactgtggcaagat agatcagtta tctcggtatc tattctcccc ttcctaatgt 38520 agaagctctgatttttaact gggcagatga ctacatggat tcaaggtatt ttcctagtgt 38580 ctcttgaaattaggtgtggt attgtgacca aattctagcc aaaaagatgt tgaacagaag 38640 tggtatatgcagcttctagg aagtgttctt aagggggaag gcgtcatccc ccacctttcc 38700 tccttcctgcactctggaat gtggaccaga tggctggagt aggagctgcc acctggggcc 38760 agaaaatgatcttaggcgtg aaggtcaagt aaggcaggac aagagagaag gagcctgcct 38820 ccccgaggctgtgaagagcc atgccagtac tgtcacctgg gagaattaaa ctggtcaagt 38880 gcctgttcgtttagggtttc atcacttgaa ggtgaccgga attctaactg atggagggag 38940 gggcccagagcaccctggag aaaggagcaa gggacgtatg gctggtggtg tctgcatgga 39000 agggtttcacagaggaaatg atgcttgagc acattttgac ccaggtggtg gaagggaaat 39060 gtggctttgagtgacaaggt gatttcacca gctcagcctt catattacca catccctacc 39120 ctttgttagactttattgtg ccctagggga tgaggctgaa ggagaccaaa aagcatct 39178 12 684 DNA H.sapiens misc_feature 32 n = A,T,C or G 12 tctttgggca gacatcaacaccaaagaaca tnctacccac tattctgcga ttgatggtac 60 cctgtttgcc aatgtccgcttcaatgtggc caattctctc cagaagatag tccccatcct 120 gaacaacagc accttgcagattgaagtcaa gcccatccta gagaagctga cccatgacca 180 ggatgtgacg tcaaataactttcccaggaa ggctctgact gttctgtctc tcgcctgatg 240 ctggaagagg agcaaacactggcctctggt gtccaccctc caacccccac aagtccctct 300 ttggggagac actgggggccgttttcttgt cactccctgt gcatggtctg accccaggcc 360 ccttccccca gcacggttcctcctctcccc agcctgggaa gatgtctcac tgtccacctc 420 ccaacgggct aggggagcacggggttggac aggacagtga ccttgggagg aaggggctac 480 tccgcccacg tcagggagagatgtgagcat cccgggtcac tggatcctgc tgctgtaatg 540 ggaacccctc ccccatttacttctccacct cccgtccttc tcatcattgg tttttttttg 600 tgtgtcaact gtgccgtttttattttattc cttttatttt cccccttttc acagagaaat 660 aaaggtctag aagtagttggtcaa 684 13 20 DNA Artificial Sequence Antisense Oligonucleotide 13gcggacattg gcaaccgggt 20 14 20 DNA Artificial Sequence AntisenseOligonucleotide 14 acatgatcac attctcccga 20 15 20 DNA ArtificialSequence Antisense Oligonucleotide 15 ttggcaaccg ggtccccagc 20 16 20 DNAArtificial Sequence Antisense Oligonucleotide 16 aaacttttcc actagcttct20 17 20 DNA Artificial Sequence Antisense Oligonucleotide 17 ggacatgatcacattctccc 20 18 20 DNA Artificial Sequence Antisense Oligonucleotide 18gaggttttca cagaactctt 20 19 20 DNA Artificial Sequence AntisenseOligonucleotide 19 acaggttccg gaagtactgt 20 20 20 DNA ArtificialSequence Antisense Oligonucleotide 20 gcctggcgca gagtgggcat 20 21 20 DNAArtificial Sequence Antisense Oligonucleotide 21 aaccgggtcc ccagccatgc20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22 tcaggcgagagacagaacag 20 23 20 DNA Artificial Sequence Antisense Oligonucleotide 23cagaagagcg tagtcatgcg 20 24 20 DNA Artificial Sequence AntisenseOligonucleotide 24 tcagaccatg cacagggagt 20 25 20 DNA ArtificialSequence Antisense Oligonucleotide 25 tcccagctgt ccagccagga 20 26 20 DNAArtificial Sequence Antisense Oligonucleotide 26 ccaggccatg cacaaggagt20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27 tgggtcagcttctctaggat 20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28gttagagatg atgttcagcc 20 29 20 DNA Artificial Sequence AntisenseOligonucleotide 29 cagctgttct gccagggcca 20 30 20 DNA ArtificialSequence Antisense Oligonucleotide 30 gcggacggcc caggacttgt 20 31 20 DNAArtificial Sequence Antisense Oligonucleotide 31 ggcagtgcac gtactctggg20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32 tctggaaggcagggaccagg 20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33atgattgtgg catgggccca 20 34 20 DNA Artificial Sequence AntisenseOligonucleotide 34 agacccatga tgactgaggc 20 35 20 DNA ArtificialSequence Antisense Oligonucleotide 35 ctgggacatg atcacattct 20 36 20 DNAArtificial Sequence Antisense Oligonucleotide 36 ggaccaggtc tgtcttggtg20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37 cctttcctgtcagactgcgg 20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38acagaacagt cagagcctcc 20 39 20 DNA Artificial Sequence AntisenseOligonucleotide 39 ccggaagtac tgtcgaagtt 20 40 20 DNA ArtificialSequence Antisense Oligonucleotide 40 gacagaacag tcagagcctc 20 41 20 DNAArtificial Sequence Antisense Oligonucleotide 41 aagaggccgc aggccgaggt20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42 cagaggccaggttggagaac 20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43catgatcaca ttctcccgac 20 44 20 DNA Artificial Sequence AntisenseOligonucleotide 44 cccacagacc tcagacagca 20 45 20 DNA ArtificialSequence Antisense Oligonucleotide 45 cagtgtttgc tcctcttcca 20 46 20 DNAArtificial Sequence Antisense Oligonucleotide 46 agggaccagg tctgtcttgg20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47 gcaagatctgggacatgatc 20 48 20 DNA Artificial Sequence Antisense Oligonucleotide 48ttggttggca tcggacacca 20 49 20 DNA Artificial Sequence AntisenseOligonucleotide 49 actcccccat ggagcggcgg 20 50 20 DNA ArtificialSequence Antisense Oligonucleotide 50 tgccaaggtg ctggagctgg 20 51 20 DNAArtificial Sequence Antisense Oligonucleotide 51 gggccggccg tccaagctgg20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52 ggccgccgccatcttggctc 20 53 20 DNA Artificial Sequence Antisense Oligonucleotide 53gagttcgtct atgagcaccg 20 54 20 DNA Artificial Sequence AntisenseOligonucleotide 54 cagcttcttg atgctgttga 20 55 20 DNA ArtificialSequence Antisense Oligonucleotide 55 aaaggcagaa gctcacttcg 20 56 20 DNAArtificial Sequence Antisense Oligonucleotide 56 agggtagtga aggttcccag20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57 cagcggtggcagcaggcagt 20 58 20 DNA Artificial Sequence Antisense Oligonucleotide 58ctcgtgtgag atggcccgta 20 59 20 DNA Artificial Sequence AntisenseOligonucleotide 59 gggaggtgaa ccagtcgccg 20 60 20 DNA ArtificialSequence Antisense Oligonucleotide 60 ccttcacagc actggacact 20 61 20 DNAArtificial Sequence Antisense Oligonucleotide 61 tggcaaactc ccccagcttg20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62 gcaccgagtcctgctcgtca 20 63 20 DNA Artificial Sequence Antisense Oligonucleotide 63ctctttgacc ttgtgggagg 20 64 20 DNA Artificial Sequence AntisenseOligonucleotide 64 acaccagctc cttgatgcag 20 65 20 DNA ArtificialSequence Antisense Oligonucleotide 65 ccagggcaga cttgacatgt 20 66 20 DNAArtificial Sequence Antisense Oligonucleotide 66 aggtgctcga tggtgttgtc20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67 acctcgttcacacagtccag 20 68 20 DNA Artificial Sequence Antisense Oligonucleotide 68aggagttaag tttctcatca 20 69 20 DNA Artificial Sequence AntisenseOligonucleotide 69 tagcatgtgc ttggtggtga 20 70 20 DNA ArtificialSequence Antisense Oligonucleotide 70 ctgcaaggtg ctgttgtcca 20 71 20 DNAArtificial Sequence Antisense Oligonucleotide 71 tcttccagca tcaggcgaga20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72 gtgagacatcttcccaggct 20 73 20 DNA Artificial Sequence Antisense Oligonucleotide 73tcggtgaatt gccacctccg 20 74 20 DNA Artificial Sequence AntisenseOligonucleotide 74 gcggccctcc cctacttgga 20 75 20 DNA ArtificialSequence Antisense Oligonucleotide 75 tgcttgagaa agatggccta 20 76 20 DNAArtificial Sequence Antisense Oligonucleotide 76 cctttgttac ctgtaaggaa20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77 ggatcatcactaggtccaag 20 78 20 DNA Artificial Sequence Antisense Oligonucleotide 78agagactcac tgtcgaagtt 20 79 20 DNA Artificial Sequence AntisenseOligonucleotide 79 taaagatctg ttaccagaag 20 80 20 DNA ArtificialSequence Antisense Oligonucleotide 80 ctttctggag ctgcagacag 20 81 20 DNAArtificial Sequence Antisense Oligonucleotide 81 gtgcccttac ctcatccttc20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82 tcccattacagcagcaggat 20 83 20 DNA Artificial Sequence Antisense Oligonucleotide 83accaatgatg agaaggacgg 20 84 20 DNA Artificial Sequence AntisenseOligonucleotide 84 gacctttatt tctctgtgaa 20 85 20 DNA H. sapiens 85gctggggacc cggttgccaa 20 86 20 DNA H. sapiens 86 gggagaatgt gatcatgtcc20 87 20 DNA H. sapiens 87 gcatggctgg ggacccggtt 20 88 20 DNA H. sapiens88 ctgttctgtc tctcgcctga 20 89 20 DNA H. sapiens 89 actccttgtgcatggcctgg 20 90 20 DNA H. sapiens 90 ggctgaacat catctctaac 20 91 20 DNAH. sapiens 91 gcctcagtca tcatgggtct 20 92 20 DNA H. sapiens 92agaatgtgat catgtcccag 20 93 20 DNA H. sapiens 93 caccaagaca gacctggtcc20 94 20 DNA H. sapiens 94 ccgcagtctg acaggaaagg 20 95 20 DNA H. sapiens95 gtcgggagaa tgtgatcatg 20 96 20 DNA H. sapiens 96 tggaagaggagcaaacactg 20 97 20 DNA H. sapiens 97 gatcatgtcc cagatcttgc 20 98 20 DNAH. sapiens 98 tggtgtccga tgccaaccaa 20 99 20 DNA H. sapiens 99gagccaagat ggcggcggcc 20 100 20 DNA H. sapiens 100 cggtgctcat agacgaactc20 101 20 DNA H. sapiens 101 tcaacagcat caagaagctg 20 102 20 DNA H.sapiens 102 cgaagtgagc ttctgccttt 20 103 20 DNA H. sapiens 103ctgggaacct tcactaccct 20 104 20 DNA H. sapiens 104 actgcctgct gccaccgctg20 105 20 DNA H. sapiens 105 tacgggccat ctcacacgag 20 106 20 DNA H.sapiens 106 agtgtccagt gctgtgaagg 20 107 20 DNA H. sapiens 107caagctgggg gagtttgcca 20 108 20 DNA H. sapiens 108 tgacgagcag gactcggtgc20 109 20 DNA H. sapiens 109 cctcccacaa ggtcaaagag 20 110 20 DNA H.sapiens 110 ctgcatcaag gagctggtgt 20 111 20 DNA H. sapiens 111acatgtcaag tctgccctgg 20 112 20 DNA H. sapiens 112 gacaacacca tcgagcacct20 113 20 DNA H. sapiens 113 ctggactgtg tgaacgaggt 20 114 20 DNA H.sapiens 114 tgatgagaaa cttaactcct 20 115 20 DNA H. sapiens 115tcaccaccaa gcacatgcta 20 116 20 DNA H. sapiens 116 tggacaacag caccttgcag20 117 20 DNA H. sapiens 117 tctcgcctga tgctggaaga 20 118 20 DNA H.sapiens 118 agcctgggaa gatgtctcac 20 119 20 DNA H. sapiens 119taggccatct ttctcaagca 20 120 20 DNA H. sapiens 120 cttggaccta gtgatgatcc20 121 20 DNA H. sapiens 121 atcctgctgc tgtaatggga 20 122 20 DNA H.sapiens 122 ttcacagaga aataaaggtc 20

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targetedto a nucleic acid molecule encoding PPP2R1A, wherein said compoundspecifically hybridizes with said nucleic acid molecule encoding PPP2R1Aand inhibits the expression of PPP2R1A.
 2. The compound of claim 1 whichis an antisense oligonucleotide.
 3. The compound of claim 2 wherein theantisense oligonucleotide comprises at least one modifiedinternucleoside linkage.
 4. The compound of claim 3 wherein the modifiedinternucleoside linkage is a phosphorothioate linkage.
 5. The compoundof claim 2 wherein the antisense oligonucleotide comprises at least onemodified sugar moiety.
 6. The compound of claim 5 wherein the modifiedsugar moiety is a 2′-O-methoxyethyl sugar moiety.
 7. The compound ofclaim 2 wherein the antisense oligonucleotide comprises at least onemodified nucleobase.
 8. The compound of claim 7 wherein the modifiednucleobase is a 5-methylcytosine.
 9. The compound of claim 2 wherein theantisense oligonucleotide is a chimeric oligonucleotide.
 10. A compound8 to 80 nucleobases in length which specifically hybridizes with atleast an 8-nucleobase portion of a preferred target region on a nucleicacid molecule encoding PPP2R1A.
 11. A composition comprising thecompound of claim 1 and a pharmaceutically acceptable carrier ordiluent.
 12. The composition of claim 11 further comprising a colloidaldispersion system.
 13. The composition of claim 11 wherein the compoundis an antisense oligonucleotide.
 14. A method of inhibiting theexpression of PPP2R1A in cells or tissues comprising contacting saidcells or tissues with the compound of claim 1 so that expression ofPPP2R1A is inhibited.
 15. A method of treating an animal having adisease or condition associated with PPP2R1A comprising administering tosaid animal a therapeutically or prophylactically effective amount ofthe compound of claim 1 so that expression of PPP2R1A is inhibited. 16.A method of screening for an antisense compound, the method comprisingthe steps of: a. contacting a preferred target region of a nucleic acidmolecule encoding PPP2R1A with one or more candidate antisensecompounds, said candidate antisense compounds comprising at least an8-nucleobase portion which is complementary to said preferred targetregion, and b. selecting for one or more candidate antisense compoundswhich inhibit the expression of a nucleic acid molecule encodingPPP2R1A.
 17. The method of claim 15 wherein the disease or condition isa neurodegenerative disorder.
 18. The method of claim 15 wherein thedisease or condition arises from aberrant apoptosis.
 19. The method ofclaim 15 wherein the disease or condition is a hyperproliferativedisorder.
 20. The method of claim 19 wherein the hyperproliferativedisorder is cancer.